Microorganisms for the production of methacrylic acid

ABSTRACT

The invention provides a non-naturally occurring microbial organism having a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid pathway. The invention additionally provides a method for producing 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid. The method can include culturing a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid producing microbial organism expressing at least one exogenous nucleic acid encoding a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid pathway enzyme in a sufficient amount and culturing under conditions and for a sufficient period of time to produce 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid.

This application is a continuation of application Ser. No. 12/433,829,filed Apr. 30, 2009, which claims the benefit of priority of U.S.Provisional application Ser. No. 61/049,730, filed May 1, 2008, each ofwhich the entire contents are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having methacrylic acid,2-hydroxyisobutyrate and 3-hydroxyisobutyrate biosynthetic capabilities.

Methyl methacrylate is an organic compound with the formulaCH₂═C(CH₃)CO₂CH₃. This colourless liquid is the methyl ester ofmethacrylic acid (MMA) and is the monomer for the production of thetransparent plastic polymethyl methacrylate (PMMA). Methyl methacrylate(MMA) is a key intermediate chemical with a global demand in excess of4.5 billion pounds per year, much of which is converted topolyacrylates.

Most commercial producers apply an acetone cyanohydrin (ACH) route toproduce methacrylic acid (MAA), with acetone and hydrogen cyanide as rawmaterials. The intermediate cyanohydrin is converted with sulfuric acidto a sulfate ester of the methacrylamide, hydrolysis of which givesammonium bisulfate and MAA. Some producers start with an isobutylene or,equivalently, tert-butanol, which is oxidized to methacrolein, and againoxidized to methacrylic acid. MAA is then esterified with methanol toMMA.

The conventional production process, using the acetone cyanohydrinroute, involves the conversion of hydrogen cyanide (HCN) and acetone toacetone cyanohydrin, which then undergoes acid assisted hydrolysis andesterification with methanol to give MMA. Difficulties in handlingpotentially deadly HCN along with the high costs of byproduct disposal(1.2 tons of ammonium bisulfate are formed per ton of MMA) have sparkeda great deal of research aimed at cleaner and more economical processes.A number of new processes have been commercialized over the last twodecades and many more are close to commercialization. The Asahi “DirectMetha” route, which involves the oxidation of isobutylene tomethacrolein, which is then mixed with methanol, oxidized with air, andesterified to MMA, has been described as an economical process.

The principal application of methyl methacrylate is the production ofpolymethyl methacrylate acrylic plastics. Also, methyl methacrylate isused for the production of the co-polymer methylmethacrylate-butadiene-styrene (MBS), used as a modifier for PVC. Methylmethacrylate polymers and co-polymers are used for waterborne coatings,such as latex paint. Uses are also found in adhesive formulations.Contemporary applications include the use in plates that keep lightspread evenly across liquid crystal display (LCD) computer and TVscreens. Methyl methacrylate is also used to prepare corrosion casts ofanatomical organs, such as coronary arteries of the heart.

Methacrylic acid, or 2-methyl-2-propenoic acid, is a low molecularweight carboxylic acid that occurs naturally in small amounts in the oilof Roman chamomile. It is a corrosive liquid with an acrid unpleasantodor. It is soluble in warm water and miscible with most organicsolvents.

Methacrylic acid polymerizes readily upon heating or treatment with acatalytic amount of strong acid, such as HCl. The resulting polymer is aceramic-looking plastic. Methacrylic acid is used industrially in thepreparation of its esters, known collectively as methacrylates, such asmethyl methacrylate, as discussed above. The methacrylates have numeroususes, most notably in the manufacture of polymers with trade names suchas Lucite™ and Plexiglas™.

Other than MMA polymers, the other major product of this industry iscrude methacrylic acid (crude MAA, FIG. 1), which accounts for about 20percent of the total production of MMA. Crude MAA is processed intobutyl methacrylates and/or “glacial” MAA, which is highly purified crudeMAA. Glacial MAA can be used directly as a comonomer in various polymersand is also used to make a variety of small volume methacrylates. On theother hand, MAA can also be converted into MMA via esterification withmethanol.

Thus, there exists a need for alternative methods for effectivelyproducing commercial quantities of compounds such as methacrylic acid,2-hydroxyisobutyrate or 3-hydroxyisobutyrate. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides a non-naturally occurring microbial organismhaving a methacrylic acid pathway. The microbial organism contains atleast one exogenous nucleic acid encoding an enzyme in a methacrylicacid pathway. The invention additionally provides a method for producingmethacrylic acid. The method can include culturing methacrylic acidproducing microbial organism, where the microbial organism expresses atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme in a sufficient amount to produce methacrylic acid, underconditions and for a sufficient period of time to produce methacrylicacid. The invention also describes organisms and production methods forthe methacrylic acid precursors 3-hydroxyisobutyrate and2-hydroxyisobutyrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of methacrylic acid (MAA).

FIG. 2 shows an exemplary metabolic pathway from succinyl-CoA to MMA via3-hydroxyisobutyrate.

FIG. 3 shows a comparison of known enzyme-catalyzed dehydrations with apredicted transformation for the dehydration of 3-hydroxyisobutyrate.FIG. 3A shows conversion of 2-hydroxymethyl glutarate by2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-). FIG. 3B showsdehydration of malate to fumarate by fumarate hydratase (EC 4.2.1.2).FIG. 3C shows the predicted dehydration of 3-hydroxyisobutyrate to MAA.

FIG. 4 shows an exemplary succinyl-CoA to MAA pathway via3-amino-2-methylpropionate. The “lumped reaction” (steps 2-3) iscatalyzed by 1) methylmalonyl-CoA epimerase and 2) methylmalonyl-CoAreductase.

FIG. 5 shows a comparison of the known enzyme-catalyzed deamination ofaspartate to form fumarate (FIG. 5A, EC 4.3.1.1) with the predicteddeamination of 3-amino-2-methylpropionate to MAA (FIG. 5B).

FIG. 6 shows an exemplary 4-hydroxybutyryl-CoA to MAA pathway thatproceeds via 3-hydroxyisobutyrate or methacrylyl-CoA. Step 2 can becatalyzed by three alternative enzymes: 3-hydroxyisobutyryl-CoAsynthetase, 3-hydroxyisobutyryl-CoA hydrolase or 3-hydroxyisobutyryl-CoAtransferase. Similarly, step 5 can be catalyzed by three alternativeenzymes: methacrylyl-CoA synthetase, methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase.

FIG. 7 shows a comparison of enzyme candidates for catalyzing theconversion of 4-hydroxybutyryl-CoA to 3-hydroxyisobutyryl-CoA. Pathwaysencoded by candidate methylmutases: FIG. 7A, methylmalonyl-CoA mutase(MCM, EC 5.4.99.2); FIG. 7B, isobutyryl-CoA mutase (ICM, EC 5.4.99.13);and FIG. 7C, predicted transformation proposed in FIG. 6 step 1.

FIG. 8 shows an exemplary alpha-ketoglutarate to MAA pathway viathreo-3-methylaspartate.

FIG. 9 shows a comparison of known enzyme-catalyzed decarboxylationswith the predicted decarboxylation of mesaconate. FIG. 9A showstransformation from aconitate to iconitate catalyzed by aconitatedecarboxylase (EC 4.1.1.6). FIG. 9B shows decarboxylation of4-oxalocrotonate to 2-oxopentenoate by 4-oxalocrotonate decarboxylase(EC 4.1.1.77). FIG. 9C shows the predicted decarboxylation of mesaconateto form MAA.

FIG. 10 shows an exemplary alpha-ketoglutarate to MAA pathway via2-hydroxyglutarate.

FIG. 11 shows enzyme candidates for 3-methylmalate conversion tomesaconate. FIG. 11A shows transformation from 2-methylmalate tomesaconate catalyzed by 2-methylmalate dehydratase (EC 4.2.1.34). FIG.11B shows dehydration of malate to fumarate by fumarate hydratase (EC4.2.1.2). FIG. 11C shows the predicted dehydration of 3-methylmalate tomesaconate.

FIG. 12 shows exemplary metabolic pathways for the conversion ofacetyl-CoA or 4-hydroxybutyryl-CoA into MAA or 2-hydroxyisobutyrate.

FIG. 13 shows an exemplary pathway from acetyl-CoA to MAA.

FIG. 14 shows an exemplary acrylyl-CoA to MAA pathway.

FIG. 15 shows an exemplary 2-ketovalerate to MAA pathway.2-Ketoisovalerate can be produced either from valine or pyruvate. Anexemplary set of enzymes for pyruvate conversion to 2-ketoisovalerate iscomprised of acetolactate synthase, acetohydroxy acid isomeroreductase,and dihydroxyacid dehydratase.

FIG. 16 shows hypothetical production envelopes of an OptKnock-designedstrain compared to a typical non-growth-coupled production strain. Thearea to the right of the diagonal relates to a typical productionstrain, whereas the left of the diagonal represents an Optknock-designedstrain. The potential evolutionary trajectories of the OptKnock strainare fundamentally different in that they lead to a high producingphenotype. The open circles within the shaded areas represent prior togrowth selection. The circles at the apex of the shaded areas (B forOptknock, A for typical production strain) represent phenotypesfollowing growth selection.

FIG. 17 shows growth-coupled MAA and 3-hydroxyisobutyrate productioncharacteristics of the highest priority knockout strain designs (gray)compared with those of wild-type E. coli (black). A glucose uptake rateof 10 mmol/gDW/hr is assumed.

FIG. 18 shows growth-coupled MAA production characteristics of thehighest priority knockout strains (gray) for a 4-hydroxybutyryl-CoA toMAA pathway compared to those of wild-type E. coli (black). A glucoseuptake rate of 10 mmol/gDW/hr is assumed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cellsand organisms having biosynthetic production capabilities formethacrylic acid. The results described herein indicate that metabolicpathways can be designed and recombinantly engineered to achieve thebiosynthesis of methacrylic acid in Escherichia coli and other cells ororganisms. Biosynthetic production of methacrylic acid can be confirmedby construction of strains having the designed metabolic genotype. Thesemetabolically engineered cells or organisms also can be subjected toadaptive evolution to further augment methacrylic acid biosynthesis,including under conditions approaching theoretical maximum growth.

As disclosed herein, organisms and methods are provided for producing2-methacrylic acid via fermentation from a renewable sugar feedstock.Described herein are high-yielding metabolic pathways for producing MAAfrom succinyl-CoA, alpha-ketoglutarate, acetyl-CoA, or other centralmetabolic precursors. Disclosed herein are pathways, their maximumproduct and ATP yields, and candidate genes for implementation offermentative MAA production.

It is understood that pathways passing through a 3-hydroxyisobutyrateintermediate can be applied for 3-hydroxyisobutyrate production asopposed to methacrylate production if the downstream enzyme, that is, adehydratase, is omitted (see FIGS. 2 and 6). In this case, thenon-naturally occurring organism would produce 3-hydroxyisobutyrateinstead of methacrylate. The non-naturally occurring organism couldalternatively produce a mixture of 3-hydroxyisobutyate and methacrylate.The maximum molar yields of ATP and product will be unchanged regardlessof whether methacrylate or 3-hydroxyisobutyrate is produced. It is alsounderstood that the pathway passing through a 2-hydroxyisobutyryl-CoAintermediate can be applied for 2-hydroxyisobutyrate production asopposed to methacrylate production if the downstream enzyme, that is, adehydratase, is omitted and a 2-hydroxyisobutyryl-CoA transferase,synthetase, or hydrolase is applied (see FIG. 12). In this case, thenon-naturally occurring organism would produce 2-hydroxyisobutyrateinstead of methacrylate. The non-naturally occurring organism couldalternatively produce a mixture of 2-hydroxyisobutyate and methacrylate.The maximum molar yields of ATP and production will be unchangedregardless of whether methacrylate or 2-hydroxyisobutyrate is produced.

It is further understood that, if desired, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid expressed by a microbial organism of theinvention can be chemically converted to methacrylic acid. For example,3-hydroxyisobutyric acid, or β-hydroxyisobutyric acid, can be dehydratedto form methacrylic acid as described, for example, in U.S. Pat. No.7,186,856. 2-Hydroxyisobutyric acid, or α-hydroxyisobutyric acid, canalso be dehydrated to form methacrylic acid as described in U.S. Pat.No. 3,666,805 and U.S. Pat. No. 5,225,594.

Two pathways originating from succinyl-CoA, described in Examples I andIII, and two pathways originating from 4-hydroxybutyryl-CoA, describedin Example V and XIX, provide high yields under anaerobic conditions(1.33 mol/mol glucose), favorable energetics and the availability ofsuitable enzyme candidates. The maximum theoretical yield ofmethacrylate starting from glucose as a raw material is 1.33 mol/molglucose as shown below:

C₆H₁₂O₆→1.33C₄H₆O₂+0.67CO₂+2H₂O

Three additional pathways, described in Examples VII, IX and XI, arehigh-yielding and energetically favorable under aerobic conditions.These pathways originate from alpha-ketoglutarate (Examples VII and IX)or acetyl-CoA (Example XI) as a starting material.

Three additional pathways, described in Examples XIII-XV, provide loweryields. The alternate acetyl-CoA pathway (Example XIII) is high-yieldingunder aerobic conditions but is lengthy, involving a minimum of sevenenzymatic steps. The acrylyl-CoA pathway (Example XIV) is high-yieldingunder anaerobic and aerobic conditions, but has the disadvantages ofunfavorable energetics, formation of a toxic intermediate (acrylyl-CoA),and a high susceptibility to the secretion of fermentation byproducts.The 2-ketoisovalerate pathway is high-yielding under aerobic conditionsbut also has the disadvantage of producing a potentially toxicintermediate (MAA-CoA) (Example XV).

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes within a methacrylic acidbiosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, “methacrylic acid,” having the chemical formulaCH₂═C(CH₃)CO₂ (see FIG. 1) (IUPAC name 2-methyl-2-propenoic acid), isthe acid form of methacrylate, and it is understood that methacrylicacid and methacrylate can be used interchangebly throughout to refer tothe compound in any of its neutral or ionized forms, including any saltforms thereof. It is understood by those skilled understand that thespecific form will depend on the pH. Similarly, it is understood that2-hydroxyisobutyrate and 2-hydroxyisobutyric acid can be usedinterchangebly throughout to refer to the compound in any of its neutralor ionized forms, including any salt forms thereof. Further,3-hydroxyisobutyrate and 3-hydroxyisobutyric acid can be usedinterchangebly throughout to refer to the compound in any of its neutralor ionized forms, including any salt forms thereof.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having methacrylic acidbiosynthetic capability, those skilled in the art will understand withapplying the teaching and guidance provided herein to a particularspecies that the identification of metabolic modifications can includeidentification and inclusion or inactivation of orthologs. To the extentthat paralogs and/or nonorthologous gene displacements are present inthe referenced microorganism that encode an enzyme catalyzing a similaror substantially similar metabolic reaction, those skilled in the artalso can utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

As disclosed herein, high-yielding metabolic pathways for 2-methacrylicacid (MAA) synthesis using glucose/sucrose as a carbon substrate aredescribed. The two principal criteria for analyzing and ranking thesepathways were the maximum theoretical yields of MAA and the associatedenergetics under both aerobic and anaerobic conditions. Product andenergy yields were calculated by adding the pathways in question to anE. coli stoichiometric network in SimPheny™ that is similar to the onedescribed in Reed et al (Reed et al., Genome Biol. 4:R54 (2003)). As MAAis a charged molecule under physiological conditions, product export isassumed to be mediated by a proton-symport mechanism. This transportmechanism is not expected to encounter a thermodynamic limitation atnear neutral fermentation conditions, although it will become lessthermodynamically favorable under acidic fermentation conditions. Thereactions in the pathways and the required enzymatic activities arediscussed in the Examples.

The invention provides a non-naturally occurring microbial organismcapable of producing methacrylic acid. For example, a methacrylic acidpathway is provided in which succinyl-CoA is a precursor (see ExamplesI-IV, FIGS. 2 and 4). In one embodiment, the invention provides anon-naturally occurring microbial organism having a methacrylic acidpathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingmethylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, methylmalonyl-CoAreductase, 3-hydroxyisobutyrate dehydrogenase and 3-hydroxyisobutyratedehydratase (see Examples I and II and FIG. 2). In another embodiment,the invention provides a non-naturally occurring microbial organismhaving a methacrylic acid pathway comprising at least one exogenousnucleic acid encoding a methacrylic acid pathway enzyme expressed in asufficient amount to produce methacrylic acid, the methacrylic acidpathway comprising methylmalonyl-CoA mutase, methylmalonyl-CoAepimerase, alcohol/aldehyde dehydrogenase, and 3-hydroxyisobutyratedehydratase (see Example I). The invention additionally provides anon-naturally occurring microbial organism having a methacrylic acidpathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingmethylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, methylmalonyl-CoAreductase, 3-amino-2-methylpropionate transaminase, and3-amino-2-methylpropionate ammonia lyase (see Examples III and IV andFIG. 4).

Additionally provided is a non-naturally occurring microbial organismcontaining a methacrylic acid pathway having 4-hydroxybutyryl-CoA as aprecursor. One such embodiment is a non-naturally occurring microbialorganism having a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising 4-hydroxybutyryl-CoA mutase,3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoA hydrolaseor 3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase (see Examples V and VI and FIG. 6). Alternatively, thepathway could include 4-hydroxybutyryl-CoA mutase,3-hydroxyisobutyryl-CoA dehydratase; and methacrylyl-CoA synthetase ormethacrylyl-CoA hydrolase or methacrylyl-CoA transferase.

Further, the present invention provides a non-naturally occurringmicrobial organism containing a 3-hydroxyisobutyric acid pathway having4-hydroxybutyryl-CoA as a precursor. One such embodiment is anon-naturally occurring microbial organism having a 3-hydroxyisobutyricacid pathway comprising at least one exogenous nucleic acid encoding a3-hydroxyisobutyric acid pathway enzyme expressed in a sufficient amountto produce 3-hydroxyisobutyric acid, the 3-hydroxyisobutyric acidpathway comprising 4-hydroxybutyryl-CoA mutase; and3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoA hydrolaseor 3-hydroxyisobutyryl-CoA transferase (see Example V and FIG. 6).

The invention further provides a non-naturally occurring microbialorganism containing a methacrylic acid pathway havingalpha-ketoglutarate as a precursor. One such embodiment is anon-naturally occurring microbial organism having a methacrylic acidpathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingaspartate aminotransferase, glutamate mutase, 3-methylaspartase, andmesaconate decarboxylase (see Examples VII and VIII and FIG. 8). In yetanother embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having a methacrylicacid pathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingalpha-ketoglutarate reductase, 2-hydroxyglutamate mutase, 3-methylmalatedehydratase, and mesaconate decarboxylase (see Examples IX and X andFIG. 10).

In still another embodiment, the invention provides a non-naturallyoccurring microbial organism containing a methacrylic acid pathwayhaving acetyl-CoA as a precursor. For example, the invention provides anon-naturally occurring microbial organism having a methacrylic acidpathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingacetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase, andmethacrylyl-CoA transferase or methacrylyl-CoA hydrolase ormethacrylyl-CoA synthetase (see Examples XI and XII and FIG. 12). Inanother embodiment, the invention provides a non-naturally occurringmicrobial organism having a methacrylic acid pathway comprising at leastone exogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA mutase,2-hydroxyisobutyryl-CoA dehydratase, enoyl-CoA hydratase, and3-hydroxyisobutyryl-CoA hydrolase or 3-hydroxyisobutyryl-CoA synthetaseor 3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase (see Example XI and XII).

In still another embodiment, the invention provides a non-naturallyoccurring microbial organism containing a 2-hydroxyisobutyric acidpathway having acetyl-CoA as a precursor. For example, the inventionprovides a non-naturally occurring microbial organism having a2-hydroxyisobutyric acid pathway comprising at least one exogenousnucleic acid encoding a 2-hydroxyisobutyric acid pathway enzymeexpressed in a sufficient amount to produce 2-hydroxyisobutyric acid,the 2-hydroxyisobutyric acid pathway comprising acetoacetyl-CoAthiolase; acetoacetyl-CoA reductase; 3-hydroxybutyryl-CoA mutase; and2-hydroxyisobutyryl-CoA hydrolase or 2-hydroxyisobutyryl-CoA synthetaseor 2-hydroxyisobutyryl-CoA transferase (see Examples XI and FIG. 12).

In further embodiments, the invention provides non-naturally occurringmicrobial organisms containing a methacrylic acid or 2-hydroxyisobutyricacid pathway having 4-hydroxybutyryl-CoA as a precursor. For example,the invention provides a non-naturally occurring microbial organismhaving a methacrylic acid pathway comprising at least one exogenousnucleic acid encoding a methacrylic acid pathway enzyme expressed in asufficient amount to produce methacrylic acid, the methacrylic acidpathway comprising 4-hydroxybutyryl-CoA dehydratase; vinylacetyl-CoAΔ-isomerase; crotonase; 3-hydroxybutyryl-CoA mutase;2-hydroxyisobutyryl-CoA dehydratase; and methacrylyl-CoA hydrolase ormethacrylyl-CoA synthetase or methacrylyl-CoA transferase (see ExampleXVIII and FIG. 12). Further, the invention provides a non-naturallyoccurring microbial organism having a 2-hydroxyisobutyric acid pathwaycomprising at least one exogenous nucleic acid encoding a2-hydroxyisobutyric acid pathway enzyme expressed in a sufficient amountto produce 2-hydroxyisobutyric acid, the 2-hydroxyisobutyric acidpathway comprising 4-hydroxybutyryl-CoA dehydratase; vinylacetyl-CoAΔ-isomerase; crotonase; 3-hydroxybutyryl-CoA mutase; and2-hydroxyisobutyryl-CoA hydrolase or 2-hydroxyisobutyryl-CoA synthetaseor 2-hydroxyisobutyryl-CoA transferase (see Examples XVIII and FIG. 12).

In yet another embodiment, the invention provides a non-naturallyoccurring microbial organism having a methacrylic acid pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, the methacrylic acid pathway comprisingacetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, crotonase,butyryl-CoA dehydrogenase, isobutyryl-CoA mutase, isobutyryl-CoAdehydrogenase, and methacrylyl-CoA synthetase or methacrylyl-CoAhydrolase or methacrylyl-CoA transferase (see Example XIII and FIG. 13).

The invention further provides a non-naturally occurring microbialorganism containing a methacrylic acid pathway having pyruvate as aprecursor. For example, the invention provides a non-naturally occurringmicrobial organism having a methacrylic acid pathway comprising at leastone exogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising lactate dehydrogenase, lactate-CoAtransferase, lactoyl-CoA dehydratase, acyl-CoA dehydrogenase,propionyl-CoA carboxylase, methylmalonyl-CoA reductase,3-hydroxyisobutyrate dehydrogenase, and 3-hydroxyisobutyrate dehydratase(see Example XIV and FIG. 14).

Also provided by the invention is a non-naturally occurring microbialorganism containing a methacrylic acid pathway having 2-ketoisovalerateas a precursor. For example, the invention provides a non-naturallyoccurring microbial organism having a methacrylic acid pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, the methacrylic acid pathway comprising valineaminotransferase, 2-ketoisovalerate dehydrogenase, isobutyryl-CoAdehydrogenase, and methacrylyl-CoA synthetase or methacrylyl-CoAhydrolase or methacrylyl-CoA transferase (see Example XV and FIG. 15).Such a methacrylic acid pathway can further contain valineaminotransferase, which converst valine to 2-ketoisovalerate (FIG. 15).In addition, such a methacrylic acid pathway can further contain enzymesthat convert pyruvate to 2-ketoisovalerate (FIG. 15), such asacetolactate synthase, acetohydroxy acid isomeroreductase anddihydroxy-acid dehydratase (see Example XV).

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a 2-hydroxyisobutyric acid,3-hydroxyisobutyric acid or methacrylic acid pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding a protein or enzyme that converts asubstrate to a product. Such a pathway can be, for example, succinyl-CoAto methylmalonyl-CoA, methylmalonyl-CoA to methylmalonate semialdehyde,methylmalonate semialdehyde to 3-hydroxyisobutyrate for a succinyl-CoAto 3-hydroxyisobutyrate pathway, and additionally 3-hydroxyisobutyrateto methacrylic acid for a succinyl-CoA to methacrylic acid pathway (seeFIG. 2). Additionally, such a pathway can be, for example, succinyl-CoAto methylmalonyl-CoA, methylmalonyl-CoA to methylmalonate semialdehyde,methylmalonate semialdehyde to 3-amino-2-methylpriopionate, and3-amino-2-methylpriopionate to methacrylic acid for an alternativesuccinyl-CoA to methacrylic acid pathway (see FIG. 4).

In another embodiment, such a pathway can be, for example,4-hydroxybutyryl-CoA to 3-hydroxyisobutyryl-CoA, 3-hydroxyisobutyryl-CoAto 3-hydroxisobutyrate for a 4-hydroxybutyryl-CoA to 3-hydroxisobutyratepathway, and additionally 3-hydroxyisobutyrate to methacrylic acid for a4-hydroxybutyryl-CoA to methacrylic acid pathway (see FIG. 6). Further,such a pathway can be, for example, alpha-ketoglutarate to glutamate,glutamate to threo-3-methylaspartate, threo-3-methylaspartate tomesaconate, mesaconate to methacrylic acid for an alpha-ketoglutarate tomethacrylic acid pathway (FIG. 8). Also, such a pathway can be, forexample, alpha-ketoglutarate to 2-hydroxyglutarate, 2-hydroxyglutarateto 3-methylmalate, 3-methylmalate to mesaconate, and mesaconate tomethacrylic acid for an alpha-ketoglutarate to methacrylic acid pathway(FIG. 10).

In still another embodiment, such a pathway can be, for example,acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutyryl-CoA,3-hydroxybutyryl-CoA to 2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyryl-CoAto methacrylyl-CoA, and methacrylyl-CoA to methacrylic acid for anacetyl-CoA to methacrylic acid pathway (FIG. 12). Also, such a pathwaycan be, for example, 4-hydroxybutyryl-CoA to vinylacetyl-CoA,vinylacetyl-CoA to crotonyl-CoA, crotonyl-CoA to 3-hydroxybutyryl-CoA,3-hydroxybutyryl-CoA to 2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyryl-CoAto methacrylyl-CoA, and methacrylyl-CoA to methacrylic acid for a4-hydroxybutyryl-CoA to methacrylic acid pathway (FIG. 12).

In yet another embodiment, such a pathway can be, for example,acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutyryl-CoA,3-hydroxybutyryl-CoA to 2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyryl-CoAto 2-hydroxyisobutyric acid for an acetyl-CoA to 2-hydroxyisobutyricacid pathway (FIG. 12). Also, such a pathway can be, for example,4-hydroxybutyryl-CoA to vinylacetyl-CoA, vinylacetyl-CoA tocrotonyl-CoA, crotonyl-CoA to 3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoAto 2-hydroxyisobutyryl-CoA, and 2-hydroxyisobutyryl-CoA to2-hydroxyisobutyric acid for 4-hydroxybutyryl-CoA to 2-hydroxyisobutyricacid pathway (FIG. 12).

In another embodiment, such a pathway can be, for example, acetyl-CoA toacetoactyl-CoA, acetoactyl-CoA to 3-hydroxybutyryl-CoA,3-hydroxybutyryl-CoA to crotonyl-CoA, crotonyl-CoA to butyryl-CoA,butyryl-CoA to isobutyryl-CoA, isobutyryl-CoA to methacrylyl-CoA, andmethacrylyl-CoA to methacrylic acid (FIG. 13). Additionally, such apathway can be, for example, pyruvate to lactate, lactate tolactoyl-CoA, lactoyl-CoA to acrylyl-CoA, acrylyl-CoA to propionyl-CoA,propionyl-CoA to methylmalonyl-CoA, and methylmalonyl-CoA to methacrylicacid (FIG. 14). Also, such a pathway can be, for example, pyruvate to2-ketoisovalerate, 2-ketoisovalerate to isobutyryl-CoA, isobutyryl-CoAto methacrylyl-CoA, and methacrylyl-CoA to methacrylic acid for apyruvate to methacrylic acid pathway (FIG. 15). Alternatively, such apathway can be, for example, valine to 2-ketoisovalerate,2-ketoisovalerate to isobutyryl-CoA, isobutyryl-CoA to methacrylyl-CoA,and methacrylyl-CoA to methacrylic acid for a valine to methacrylic acidpathway (FIG. 15). Thus, the invention provides a non-naturallyoccurring microbial organism containing at least one exogenous nucleicacid encoding an enzyme or protein that converts the substrates andproducts of a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid ormethacryl acid pathway, such as that shown in FIGS. 2, 4, 6, 8, 10, and12-15.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing the referenced metabolic reaction,reactant or product. Unless otherwise expressly stated herein, thoseskilled in the art will understand that reference to a reaction alsoconstitutes reference to the reactants and products of the reaction.Similarly, unless otherwise expressly stated herein, reference to areactant or product also references the reaction, and reference to anyof these metabolic constituents also references the gene or genesencoding the enzymes that catalyze the referenced reaction, reactant orproduct. Likewise, given the well known fields of metabolicbiochemistry, enzymology and genomics, reference herein to a gene orencoding nucleic acid also constitutes a reference to the correspondingencoded enzyme and the reaction it catalyzes as well as the reactantsand products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes participating in one or more methacrylic acidbiosynthetic pathways. Similarly, non-naturally occurring organisms ofthe invention can be produced by introducing expressible nucleic acidsencoding one or more of the enzymes participating in one or more3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid pathways. Dependingon the host microbial organism chosen for biosynthesis, nucleic acidsfor some or all of a particular methacrylic acid, 3-hydroxyisobutyricacid or 2-hydroxyisobutyric acid biosynthetic pathway can be expressed.For example, if a chosen host is deficient in one or more enzymes for adesired biosynthetic pathway, then expressible nucleic acids for thedeficient enzyme(s) are introduced into the host for subsequentexogenous expression. Alternatively, if the chosen host exhibitsendogenous expression of some pathway genes, but is deficient in others,then an encoding nucleic acid is needed for the deficient enzyme(s) toachieve methacrylic acid, 3-hydroxyisobutyric acid, or2-hydroxyisobutyric acid biosynthesis. Thus, a non-naturally occurringmicrobial organism of the invention can be produced by introducingexogenous enzyme activities to obtain a desired biosynthetic pathway ora desired biosynthetic pathway can be obtained by introducing one ormore exogenous enzyme activities that, together with one or moreendogenous enzymes, produces a desired product such as methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid.

Depending on the methacrylic acid biosynthetic pathway constituents of aselected host microbial organism, the non-naturally occurring microbialorganisms of the invention will include at least one exogenouslyexpressed methacrylic acid pathway-encoding nucleic acid and up to allencoding nucleic acids for one or more methacrylic acid biosyntheticpathways. For example, methacrylic acid biosynthesis can be establishedin a host deficient in a pathway enzyme through exogenous expression ofthe corresponding encoding nucleic acid. In a host deficient in allenzymes of a methacrylic acid pathway, exogenous expression of allenzyme in the pathway can be included, although it is understood thatall enzymes of a pathway can be expressed even if the host contains atleast one of the pathway enzymes. Similarly, depending on the3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid biosyntheticpathway constituents of a selected host microbial organism, thenon-naturally occurring microbial organisms of the invention willinclude at least one exogenously expressed 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid pathway-encoding nucleic acid and up to allencoding nucleic acids for one or more 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid biosynthetic pathways, respectively.

For example, exogenous expression of all enzymes in a pathway forproduction of methacrylic acid can be included. For example, all enzymesin a pathway for production of methacrylic acid can be included, such asmethylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, methylmalonyl-CoAreductase, 3-hydroxyisobutyrate dehydrogenase and 3-hydroxyisobutyratedehydratase. Another example of enzymes in a methacrylic acid pathwayincludes methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase,alcohol/aldehyde dehydrogenase, and 3-hydroxyisobutyrate dehydratase. Afurther example of enzymes in a methacrylic acid pathway includesmethylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, methylmalonyl-CoAreductase, 3-amino-2-methylpropionate transaminase, and3-amino-2-methylpropionate ammonia lyase. In still another example ofenzymes in a methacrylic acid pathway includes 4-hydroxybutyryl-CoAmutase, 3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoAhydrolase or 3-hydroxyisobutyryl-CoA transferase, and3-hydroxyisobutyrate dehydratase. Also, an example of enzymes in amethacrylic acid pathway includes aspartate aminotransferase, glutamatemutase, 3-methylaspartase, and mesaconate decarboxylase. Yet anotherexample of enzymes in a methacrylic acid pathway includesalpha-ketoglutarate reductase, 2-hydroxyglutamate mutase, 3-methylmalatedehydratase, and mesaconate decarboxylase. A further example of enzymesin a methacrylic acid pathway includes acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA mutase,2-hydroxyisobutyryl-CoA dehydratase, and methacrylyl-CoA transferase ormethacrylyl-CoA hydrolase or methacrylyl-CoA synthetase. Still anotherexample of enzymes in a methacrylic acid pathway includesacetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase,enoyl-CoA hydratase, and 3-hydroxyisobutyryl-CoA hydrolase or3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoAtransferase, and 3-hydroxyisobutyrate dehydratase. It is understood thatthese and any of the methacrylic acid pathways disclosed herein can beutilized in a microbial organism to generate a methacrylic acidproducing microbial organism.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel themethacrylic acid pathway deficiencies of the selected host microbialorganism. Therefore, a non-naturally occurring microbial organism of theinvention can have one, two, three, four, and so forth, up to allnucleic acids encoding the above enzymes constituting a methacrylic acidbiosynthetic pathway, as disclosed herein. In some embodiments, thenon-naturally occurring microbial organisms also can include othergenetic modifications that facilitate or optimize methacrylic acidbiosynthesis or that confer other useful functions onto the hostmicrobial organism. One such other functionality can include, forexample, augmentation of the synthesis of one or more of the methacrylicacid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid pathwayprecursors. Exemplary methacrylic acid pathway precursors include, butare not limited to, succinyl-CoA, 4-hydroxybutyryl-CoA,alpha-ketoglutarate, acetyl-CoA, pyruvate, and 2-ketoisovalerate.

Generally, a host microbial organism is selected such that it producesthe precursor of a methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid pathway, either as a naturally producedmolecule or as an engineered product that either provides de novoproduction of a desired precursor or increased production of a precursornaturally produced by the host microbial organism. For example,succinyl-CoA, alpha-ketoglutarate, acetyl-CoA, and pyruvate are producednaturally in a host organism such as E. coli during glucose, fatty acidand amino acid metabolism and as components of the TCA cycle. A hostorganism can be engineered to increase production of a precursor, asdisclosed herein. Such engineered microorganisms have been describedpreviously (see, for example, U.S. publication 2007/0111294). Inaddition, a microbial organism that has been engineered to produce adesired precursor can be used as a host organism, for example, amicroorganism engineered to produce 4-hydroxybutyryl-CoA (see, forexample, U.S. application Ser. No. 12/049,256, filed Mar. 14, 2008), asdisclosed herein. Such host organisms can be further engineered toexpress enzymes of a methacrylic acid 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid. In this specific embodiment it can be usefulto increase the synthesis or accumulation of a methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid pathway product to,for example, drive methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid pathway reactions toward methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid production.Increased synthesis or accumulation can be accomplished by, for example,overexpression of nucleic acids encoding one or more of theabove-described methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid pathway enzymes. Over expression of themethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acidpathway enzyme or enzymes can occur, for example, through exogenousexpression of the endogenous gene or genes, or through exogenousexpression of the heterologous gene or genes. Therefore, naturallyoccurring organisms can be readily generated to be non-naturallyoccurring microbial organisms of the invention, for example, producingmethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid,through overexpression of one, two, three, four, five, and so forth,depending on the methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid pathway, that is, up to including all nucleicacids encoding methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid biosynthetic pathway enzymes. In addition, anon-naturally occurring organism can be generated by mutagenesis of anendogenous gene that results in an increase in activity of an enzyme inthe methacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyricacid biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid biosynthetic pathway onto the microbialorganism. Alternatively, encoding nucleic acids can be introduced toproduce an intermediate microbial organism having the biosyntheticcapability to catalyze some of the required reactions to confermethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acidbiosynthetic capability. For example, a non-naturally occurringmicrobial organism having a methacrylic acid biosynthetic pathway cancomprise at least two exogenous nucleic acids encoding desired enzymes.One exemplary combination includes the combination of methylmalonyl-CoAmutase and methylmalonyl-CoA epimerase; or methylmalonyl-CoA mutase andmethylmalonyl-CoA reductase; 3-hydroxyisobutyrate dehydrogenase and3-hydroxyisobutyrate dehydratase, and the like. In another exemplarypathway, a combination can include 4-hydroxybutyryl-CoA mutase and3-hydroxyisobutyryl-CoA transferase; 3-hydroxyisobutyryl-CoA synthetaseand 3-hydroxyisobutyrate dehydratase; 4-hydroxybutyryl-CoA mutase and3-hydroxyisobutyryl-CoA synthetase, and so forth. Thus, it is understoodthat any combination of two or more enzymes of a biosynthetic pathwaycan be included in a non-naturally occurring microbial organism of theinvention.

Similarly, it is understood that any combination of three or moreenzymes of a biosynthetic pathway can be included in a non-naturallyoccurring microbial organism of the invention, for example,methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, andmethylmalonyl-CoA reductase; methylmalonyl-CoA epimerase,3-amino-2-methylpropionate transaminase, and 3-amino-2-methylpropionateammonia lyase, and so forth. In another example, the combination can bealpha-ketoglutarate reductase, 3-methylmalate dehydratase, andmesaconate decarboxylase; 2-hydroxyglutamate mutase, 3-methylmalatedehydratase, and mesaconate decarboxylase, and so forth, as desired, solong as the combination of enzymes of the desired biosynthetic pathwayresults in production of the corresponding desired product. Similarly,any combination of four, five, six or more enzymes, depending on thedesired pathway, of a biosynthetic pathway as disclosed herein can beincluded in a non-naturally occurring microbial organism of theinvention, as desired, so long as the combination of enzymes of thedesired biosynthetic pathway results in production of the correspondingdesired product.

In addition to the biosynthesis of methacrylic acid, 3-hydroxyisobutyricacid or 2-hydroxyisobutyric acid as described herein, the non-naturallyoccurring microbial organisms and methods of the invention also can beutilized in various combinations with each other and with othermicrobial organisms and methods well known in the art to achieve productbiosynthesis by other routes. For example, one alternative to producemethacrylic acid other than use of the methacrylic acid producers isthrough addition of another microbial organism capable of converting amethacrylic acid pathway intermediate to methacrylic acid. One suchprocedure includes, for example, the fermentation of a microbialorganism that produces a methacrylic acid pathway intermediate. Themethacrylic acid pathway intermediate can then be used as a substratefor a second microbial organism that converts the methacrylic acidpathway intermediate to methacrylic acid. The methacrylic acid pathwayintermediate can be added directly to another culture of the secondorganism or the original culture of the methacrylic acid pathwayintermediate producers can be depleted of these microbial organisms by,for example, cell separation, and then subsequent addition of the secondorganism to the fermentation broth can be utilized to produce the finalproduct without intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid. In theseembodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis ofmethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acidcan be accomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, methacrylicacid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid also can bebiosynthetically produced from microbial organisms through co-culture orco-fermentation using two organisms in the same vessel, where the firstmicrobial organism produces a methacrylic acid, 3-hydroxyisobutyric acidor 2-hydroxyisobutyric acid pathway intermediate and the secondmicrobial organism converts the intermediate to methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid. For example,fermentation to form 3-hydroxyisobutyric acid can be combined with apurification scheme to yield methyl methacrylate (see WO 2002/090312).

Sources of encoding nucleic acids for a methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid pathway enzyme caninclude, for example, any species where the encoded gene product iscapable of catalyzing the referenced reaction. Such species include bothprokaryotic and eukaryotic organisms including, but not limited to,bacteria, including archaea and eubacteria, and eukaryotes, includingyeast, plant, insect, animal, and mammal, including human. Exemplaryspecies for such sources include, for example, Escherichia coli, Homosapiens, Propionibacterium fredenreichii, Methylobacterium extorquens,Shigella flexneri, Salmonella enterica, Yersinia frederiksenii,Propionibacterium acnes, Rattus norvegicus, Caenorhabditis elegans,Bacillus cereus, Acinetobacter calcoaceticus, Acinetobacter baylyi,Acinetobacter sp., Clostridium kluyveri, Pseudomonas sp., Thermusthermophilus, Pseudomonas aeruginosa, Pseudomonas putida, Oryctolaguscuniculus, Clostridium acetobutylicum, Leuconostoc mesenteroides,Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis,Natranaerobius thermophilus, Campylobacter jejuni, Arabidopsis thaliana,Corynebacterium glutamicum, Sus scrofa, Bacillus subtilus, Pseudomonasfluorescens, Serratia marcescens, Streptomyces coelicolor, Methylibiumpetroleiphilum, Streptomyces cinnamonensis, Streptomyces avermitilis,Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum,Saccharomyces cerevisiae, Clostridium cochlearium, Clostridiumtetanomorphum, Clostridium tetani, Citrobacter amalonaticus, Ralstoniaeutropha, Mus musculus, Bos taurus, Fusobacterium nucleatum, Morganellamorganii, Clostridium pasteurianum, Rhodobacter sphaeroides,Xanthobacter autotrophicus, Clostridium propionicum, Megasphaeraelsdenii, Aspergillus terreus, Candida, Sulfolobus tokodaii,Metallosphaera sedula, Chloroflexus aurantiacus, Clostridiumsaccharoperbutylacetonicum, Acidaminococcus fermentans, Helicobacterpylori, as well as other exemplary species disclosed herein or availableas source organisms for corresponding genes. However, with the completegenome sequence available for now more than 550 species (with more thanhalf of these available on public databases such as the NCBI), including395 microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisitemethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acidbiosynthetic activity for one or more genes in related or distantspecies, including for example, homologues, orthologs, paralogs andnonorthologous gene displacements of known genes, and the interchange ofgenetic alterations between organisms is routine and well known in theart. Accordingly, the metabolic alterations enabling biosynthesis ofmethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric aciddescribed herein with reference to a particular organism such as E. colican be readily applied to other microorganisms, including prokaryoticand eukaryotic organisms alike. Given the teachings and guidanceprovided herein, those skilled in the art will know that a metabolicalteration exemplified in one organism can be applied equally to otherorganisms.

In some instances, such as when an alternative methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid biosyntheticpathway exists in an unrelated species, methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesizemethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger andPichia pastoris. E. coli is a particularly useful host organisms sinceit is a well characterized microbial organism suitable for geneticengineering. Other particularly useful host organisms include yeast suchas Saccharomyces cerevisiae.

Methods for constructing and testing the expression levels of anon-naturally occurring methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid-producing host can be performed, for example,by recombinant and detection methods well known in the art. Such methodscan be found described in, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory,New York (2001); and Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production ofmethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acidcan be introduced stably or transiently into a host cell usingtechniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore methacrylic acid biosynthetic pathway encoding nucleic acids asexemplified herein operably linked to expression control sequencesfunctional in the host organism. Expression vectors applicable for usein the microbial host organisms of the invention include, for example,plasmids, phage vectors, viral vectors, episomes and artificialchromosomes, including vectors and selection sequences or markersoperable for stable integration in to a host chromosome. Additionally,the expression vectors can include one or more selectable marker genesand appropriate expression control sequences. Selectable marker genesalso can be included that, for example, provide resistance toantibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

The invention additionally provides methods for producing a desiredproduct such as methacrylic acid. In one embodiment, the inventionprovides a method for producing methacrylic acid, comprising culturing anon-naturally occurring microbial organism having a methacrylic acidpathway, the pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, under conditions and for asufficient period of time to produce metharcylic acid, the methacrylicacid pathway comprising methylmalonyl-CoA mutase, methylmalonyl-CoAepimerase, methylmalonyl-CoA reductase, 3-hydroxyisobutyratedehydrogenase and 3-hydroxyisobutyrate dehydratase (see Examples I andII and FIG. 2). In another embodiment, the invention provides a methodfor producing methacrylic acid, comprising culturing a non-naturallyoccurring microbial organism having a methacrylic acid pathway, thepathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, under conditions and for a sufficient periodof time to produce methacrylic acid, the methacrylic acid pathwaycomprising methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase,alcohol/aldehyde dehydrogenase, and 3-hydroxyisobutyrate dehydratase(see Example I).

In yet another embodiment, the invention provides a method for producingmethacrylic acid, comprising culturing a non-naturally occurringmicrobial organism having a methacrylic acid pathway, the pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, under conditions and for a sufficient period of timeto produce methacrylic acid, the methacrylic acid pathway comprisingmethylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, methylmalonyl-CoAreductase, 3-amino-2-methylpropionate transaminase, and3-amino-2-methylpropionate ammonia lyase (see Examples III and IV andFIG. 4). Additionally provided is a method for producing methacrylicacid, comprising culturing a non-naturally occurring microbial organismhaving a methacrylic acid pathway, the pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, underconditions and for a sufficient period of time to produce methacrylicacid, the methacrylic acid pathway comprising 4-hydroxybutyryl-CoAmutase, 3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoAhydrolase or 3-hydroxyisobutyryl-CoA transferase, and3-hydroxyisobutyrate dehydratase (see Examples V and VI and FIG. 6).

Also provided is a method for producing methacrylic acid, comprisingculturing a non-naturally occurring microbial organism having amethacrylic acid pathway, the pathway comprising at least one exogenousnucleic acid encoding a methacrylic acid pathway enzyme expressed in asufficient amount to produce methacrylic acid, under conditions and fora sufficient period of time to produce methacrylic acid, the methacrylicacid pathway comprising aspartate aminotransferase, glutamate mutase,3-methylaspartase, and mesaconate decarboxylase (see Examples VII andVIII and FIG. 8). Another embodiment provides a method for producingmethacrylic acid, comprising culturing a non-naturally occurringmicrobial organism having a methacrylic acid pathway, the pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, under conditions and for a sufficient period of timeto produce methacrylic acid, the methacrylic acid pathway comprisingalpha-ketoglutarate reductase, 2-hydroxyglutamate mutase, 3-methylmalatedehydratase, and mesaconate decarboxylase (see Examples IX and X andFIG. 10).

In yet a further embodiment, the invention provides a method forproducing methacrylic acid, comprising culturing a non-naturallyoccurring microbial organism having a methacrylic acid pathway, thepathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, under conditions and for a sufficient periodof time to produce methacrylic acid, the methacrylic acid pathwaycomprising acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase, andmethacrylyl-CoA transferase or methacrylyl-CoA hydrolase ormethacrylyl-CoA synthetase (see Example XI and XII and FIG. 12). A stillfurther embodiment provides a method for producing methacrylic acid,comprising culturing a non-naturally occurring microbial organism havinga methacrylic acid pathway, the pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, underconditions and for a sufficient period of time to produce methacrylicacid, the methacrylic acid pathway comprising acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA mutase,2-hydroxyisobutyryl-CoA dehydratase, enoyl-CoA hydratase, and3-hydroxyisobutyryl-CoA hydrolase or 3-hydroxyisobutyryl-CoA synthetaseor 3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase (see Example XI and XII).

The invention additional provides a method for producing methacrylicacid, comprising culturing a non-naturally occurring microbial organismhaving a methacrylic acid pathway, the pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, underconditions and for a sufficient period of time to produce methacrylicacid, the methacrylic acid pathway comprising acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, crotonase, butyryl-CoA dehydrogenase,isobutyryl-CoA mutase, isobutyryl-CoA dehydrogenase, and methacrylyl-CoAsynthetase or methacrylyl-CoA hydrolase or methacrylyl-CoA transferase(see Example XIII and FIG. 13). Also provided method for producingmethacrylic acid, comprising culturing a non-naturally occurringmicrobial organism having a methacrylic acid pathway, the pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, under conditions and for a sufficient period of timeto produce methacrylic acid, the methacrylic acid pathway comprisinglactate dehydrogenase, lactate-CoA transferase, lactoyl-CoA dehydratase,acyl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoAreductase, 3-hydroxyisobutyrate dehydrogenase, and 3-hydroxyisobutyratedehydratase (see Example XIV and FIG. 14). Yet a further embodimentprovides a method for producing methacrylic acid, comprising culturing anon-naturally occurring microbial organism having a methacrylic acidpathway, the pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, under conditions and for asufficient period of time to produce methacrylic acid, the methacrylicacid pathway comprising valine aminotransferase, 2-ketoisovaleratedehydrogenase, isobutyryl-CoA dehydrogenase, and methacrylyl-CoAsynthetase or methacrylyl-CoA hydrolase or methacrylyl-CoA transferase(see Example XV and FIG. 15). Such a pathway can further compriseacetolactate synthase, acetohydroxy acid isomeroreductase anddihydroxy-acid dehydratase.

The invention additionally provides a method for producing3-hydroxyisobutyric acid, comprising culturing a non-naturally occurringmicrobial organism having a 3-hydroxyisobutyric acid pathway, thepathway comprising at least one exogenous nucleic acid encoding a3-hydroxyisobutyric acid pathway enzyme expressed in a sufficient amountto produce 3-hydroxyisobutyric acid, under conditions and for asufficient period of time to produce 3-hydroxyisobutyric acid, the3-hydroxyisobutyric acid pathway comprising 4-hydroxybutyryl-CoA mutase;and 3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoAhydrolase or 3-hydroxyisobutyryl-CoA transferase (see Example V and FIG.6). Also provided is a method for producing 2-hydroxyisobutyric acid,comprising culturing a non-naturally occurring microbial organism havinga 2-hydroxyisobutyric acid pathway, the pathway comprising at least oneexogenous nucleic acid encoding a 2-hydroxyisobutyric acid pathwayenzyme expressed in a sufficient amount to produce 2-hydroxyisobutyricacid, under conditions and for a sufficient period of time to produce2-hydroxyisobutyric acid, the 2-hydroxyisobutyric acid pathwaycomprising acetoacetyl-CoA thiolase; acetoacetyl-CoA reductase;3-hydroxybutyryl-CoA mutase; and 2-hydroxyisobutyryl-CoA transferase or2-hydroxyisobutyryl-CoA hydrolase or 2-hydroxyisobutyryl-CoA synthetase(see Example XI and FIG. 12).

In another embodiment, the invention provides a method for producingmethacrylic acid comprising culturing a non-naturally occurringmicrobial organism having a methacrylic acid pathway, the pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, under conditions and for a sufficient period of timeto produce methacrylic acid, the methacrylic acid pathway comprising4-hydroxybutyryl-CoA dehydratase; vinylacetyl-CoA Δ-isomerase;crotonase; 3-hydroxybutyryl-CoA mutase; 2-hydroxyisobutyryl-CoAdehydratase; and methacrylyl-CoA hydrolase or methacrylyl-CoA synthetaseor methacrylyl-CoA transferase (see Example XVIII and FIG. 12). Alsoprovided is a method for producing 2-hydroxyisobutyric acid, comprisingculturing a non-naturally occurring microbial organism having a2-hydroxyisobutyric acid pathway, the pathway comprising at least oneexogenous nucleic acid encoding a 2-hydroxyisobutyric acid pathwayenzyme expressed in a sufficient amount to produce 2-hydroxyisobutyricacid, under conditions and for a sufficient period of time to produce2-hydroxyisobutyric acid, the 2-hydroxyisobutyric acid pathwaycomprising 4-hydroxybutyryl-CoA dehydratase; vinylacetyl-CoAΔ-isomerase; crotonase; 3-hydroxybutyryl-CoA mutase; and2-hydroxyisobutyryl-CoA hydrolase or 2-hydroxyisobutyryl-CoA synthetaseor 2-hydroxyisobutyryl-CoA transferase (see Examples XVIII and FIG. 12).

Suitable purification and/or assays to test for the production ofmethacrylic acid can be performed using well known methods. Suitablereplicates such as triplicate cultures can be grown for each engineeredstrain to be tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy), or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme activities from the exogenous DNA sequencescan also be assayed using methods well known in the art.

The methacrylic acid, 2-hydroxyisobutyric acid or 3-hydroxyisobutyricacid products can be separated from other components in the cultureusing a variety of methods well known in the art. Such separationmethods include, for example, extraction procedures as well as methodsthat include continuous liquid-liquid extraction, pervaporation,membrane filtration, membrane separation, reverse osmosis,electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the methacrylic acid producers can becultured for the biosynthetic production of methacrylic acid.

For the production of methacrylic acid, 2-hydroxyisobutyric acid or3-hydroxyisobutyric acid, the recombinant strains are cultured in amedium with carbon source and other essential nutrients. It is highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic conditions can be applied byperforating the septum with a small hole for limited aeration. Exemplaryanaerobic conditions have been described previously and are well-knownin the art. Exemplary aerobic and anaerobic conditions are described,for example, in U.S. patent application Ser. No. 11/891,602, filed Aug.10, 2007. Fermentations can be performed in a batch, fed-batch orcontinuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can be, for example, any carbohydrate source which cansupply a source of carbon to the non-naturally occurring microorganism.Such sources include, for example, sugars such as glucose, xylose,arabinose, galactose, mannose, fructose and starch. Other sources ofcarbohydrate include, for example, renewable feedstocks and biomass.Exemplary types of biomasses that can be used as feedstocks in themethods of the invention include cellulosic biomass, hemicellulosicbiomass and lignin feedstocks or portions of feedstocks. Such biomassfeedstocks contain, for example, carbohydrate substrates useful ascarbon sources such as glucose, xylose, arabinose, galactose, mannose,fructose and starch. Given the teachings and guidance provided herein,those skilled in the art will understand that renewable feedstocks andbiomass other than those exemplified above also can be used forculturing the microbial organisms of the invention for the production ofmethacrylic acid.

In addition to renewable feedstocks such as those exemplified above, themethacrylic acid microbial organisms of the invention also can bemodified for growth on syngas as its source of carbon. In this specificembodiment, one or more proteins or enzymes are expressed in themethacrylic acid producing organisms to provide a metabolic pathway forutilization of syngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:

2CO₂+4H₂+nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes: cobalamide corrinoid/iron-sulfur protein,methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase,acetyl-CoA synthase disulfide reductase and hydrogenase. Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the Wood-Ljungdahl enzymes absent in the host organism.Therefore, introduction of one or more encoding nucleic acids into themicrobial organisms of the invention such that the modified organismcontains the complete Wood-Ljungdahl pathway will confer syngasutilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, methacrylic acid andany of the intermediate metabolites in the methacrylic acid pathway. Allthat is required is to engineer in one or more of the required enzymeactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of themethacrylic acid biosynthetic pathways. Accordingly, the inventionprovides a non-naturally occurring microbial organism that producesand/or secretes methacrylic acid when grown on a carbohydrate andproduces and/or secretes any of the intermediate metabolites shown inthe methacrylic acid pathway when grown on a carbohydrate. Intermediatemetabolites that can be produced and/or excreted include3-hydroxyisobutyric acid and 2-hydroxyisobutyric acid.

The methacrylic acid producing microbial organisms of the invention caninitiate synthesis from an intermediate. For example, in addition toinitiating synthesis from succinyl-CoA as a precursor, synthesis can beinitiated from an intermediate such as (R)-methylmalonyl-CoA,(S)-methylmalonyl-CoA, methylmalonate semialdehyde or3-hydroxyisobutyrate (see Example I and FIG. 2). Alternatively,synthesis can be initiated from an intermediate such as(R)-methylmalonyl-CoA, (S)-methylmalonyl-CoA, methylmalonatesemialdehyde, or 3-amino-2-methylpropionate (see Example III and FIG.4). In a pathway having 4-hydroxybutyryl-CoA as a precursor, synthesiscan be initiated from an intermediate such as 3-hydroxyisobutyryl-CoA,methacrylyl-CoA or 3-hydroxyisobutyrate (see Example V and FIG. 6).

In a methacrylic acid pathway utilizing alpha-ketoglutarate as aprecursor, synthesis can be initiated, for example, from glutamate,threo-3-methylaspartate or mesaconate (see Example VII and FIG. 8).Alternatively, synthesis can initiate from an intermediate such as2-hydroxyglutarate, 3-methylmalate or mesaconate (see Example IX andFIG. 10). In a pathway utilizing acetyl-CoA as a precursor, synthesiscan initiate, for example, from an intermediate such as acetoacetyl-CoA,3-hydroxybutyryl-CoA, 2-hydroxyisobutyryl-CoA, or methacrylyl-CoA (seeExample XI and FIG. 12). Alternatively, synthesis can be initiated froman intermediate such as acetoacetyl-CoA, 3-hydroxybutyryl-CoA,crotonyl-CoA, butyryl-CoA, isobutyryl-CoA and methacrylyl-CoA (seeExample XIII and FIG. 13).

In a methacrylic acid pathway utilizing pyruvate as a precursor,synthesis can initiate from an intermediate such as lactate,lactoyl-CoA, acrylyl-CoA, propionyl-CoA, (S)-methylmalonyl-CoA,methylmalonate semialdehyde or 3-hydroxyisobutyrate (see Example XIV andFIG. 14). In a pathway utilizing 2-ketoisovalerate as precursor,synthesis can initiate from an intermediate such as isobutyryl-CoA ormethacrylyl-CoA (see Example XV and FIG. 15). In addition, synthesis caninitiate from an intermediate in the conversion of pyruvate to2-ketoisovalerate.

In a 3-hydroxyisobutyric acid pathway utilizing 4-hydroxybutyryl-CoA asa precursor, synthesis can initiate from an intermediate such as3-hydroxyisobutyryl-CoA (see Example V and FIG. 6). In a2-hydroxyisobutyric acid pathway utilizing acetyl-CoA as a precursor,synthesis can initiate, for example, from an intermediate such asacetoacetyl-CoA, 3-hydroxybutyryl-CoA, or 2-hydroxyisobutyryl-CoA (seeExample XI and FIG. 12).

Furthermore, it is understood that additional modifications can be to amicrobial organism of the invention to increase product yield. Forexample, metabolic modeling can be employed to determine any additionalmodifications that can be made to a microbial organism having a2-hydroxyiosbutyric acid, 3-hydroxyisobutyric acid or methacrylic acidpathway to increase product yield (see Example XXV). In one embodiment,modifications can be employed to increase the production of a precursoror intermediate of a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acidor methacrylic acid pathway. As disclosed herein, modifications ofmetabolic pathways can be employed, including modification of centralmetabolic reactions and their corresponding enzymes, to increase theyield of a desired precursor, intermediate or product. For example, ithas been found that increasing the expression of several enzymes byvarious mechanisms can be utilized to increase the yield of MAA or3-hydroxyisobutyrate. Such enzymes include, but are not limited to, 1)citrate synthase and aconitase; 2) isocitrate lyase and malate synthase;3) pyruvate dehydrogenase and/or pyruvate ferredoxin oxidoreductase; and4) phosphoenolpyruvate (PEP) carboxykinase (see Example XXV). Expressionof these enzymes can be used to increase the yields of MAA or3-hydroxyisobutyrate using the pathways from succinyl-CoA or4-hydroxybutyryl-CoA.

Thus, the invention additionally provides a non-naturally occurringmicrobial organism which, in addition to containing a 2-hydroxisobutyricacid, 3-hydroxyisobutyric acid or methacrylic acid pathway, further isgenetically modified to increase the activity of at least one protein orenzyme that increases production of a precursor or intermediate of the2-hydroxisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acidproduct, wherein the increase in activity is relative to the absence ofthe genetic modification that increases the activity of the at least oneprotein or enzyme. For example, the non-naturally occurring microbialorganism can be genetically modified to increase the activity of atleast one of an enzyme selected from citrate synthase, aconitase,isocitrate lyase, malate synthase, pyruvate dehydrogenase, pyruvateferredoxin oxidoreductase and phosphoenolpyruvate carboxykinase (seeExample XXV). It is understood that the increase in activity is relativeto a microbial organism that has not been genetically modified toincrease the activity of such enzymes. For example, if the geneticmodification to increase the activity of an enzyme is introduced into amicrobial organism having a methacrylic acid pathway, then the increasein activity of the enzyme is relative to the host organism having amethacrylic acid pathway but in the absence of the genetic modification.It is understood that such genetic modifications include, but are notlimited to, introducing an exogenous nucleic acid encoding a homologous(native) or heterologous sequence of a protein or enzyme whose activityis to be increased, either by chromosomal integration or contained on aplasmid. For example, a heterologous sequence from an organism having adesirable property that increases the activity of the protein or enzymecan be introduced, or an increased copy number of the endogenous genecan be introduced into the organism. In addition, the promoter of theendogenous gene can be replaced with a more active promoter or thenative promoter can be genetically modified with mutations to increaseexpression and therefore activity of the protein or enzyme. Such areplacement or other genetic modification of the promoter can result ineither a constitutive or inducible promoter. Additionally, a repressorof the endogenous gene can be decreased, for example, by knocking outthe repressor with a gene disruption or genetically modifying itspromoter to decrease expression. Thus, these and other geneticmodifications disclosed herein and known in the art can be used toincrease the activity of a desired protein or enzyme.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a methacrylicacid pathway enzyme in sufficient amounts to produce methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid. It is understoodthat the microbial organisms of the invention are cultured underconditions sufficient to produce methacrylic acid, 3-hydroxyisobutyricacid or 2-hydroxyisobutyric acid. Following the teachings and guidanceprovided herein, the non-naturally occurring microbial organisms of theinvention can achieve biosynthesis of methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid resulting inintracellular concentrations between about 0.1-200 mM or more.Generally, the intracellular concentration of methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid is between about3-150 mM, particularly between about 5-125 mM and more particularlybetween about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, ormore. Intracellular concentrations between and above each of theseexemplary ranges also can be achieved from the non-naturally occurringmicrobial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007. Any of theseconditions can be employed with the non-naturally occurring microbialorganisms as well as other anaerobic conditions well known in the art.Under such anaerobic conditions, the methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid producers cansynthesize methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid, respectively, at intracellular concentrationsof 5-10 mM or more as well as all other concentrations exemplifiedherein. It is understood that, even though the above description refersto intracellular concentrations, methacrylic acid, 3-hydroxyisobutyricacid or 2-hydroxyisobutyric acid producing microbial organisms canproduce methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid intracellularly and/or secrete the product intothe culture medium.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid includes anaerobic culture or fermentationconditions. In certain embodiments, the non-naturally occurringmicrobial organisms of the invention can be sustained, cultured orfermented under anaerobic or substantially anaerobic conditions.Briefly, anaerobic conditions refers to an environment devoid of oxygen.Substantially anaerobic conditions include, for example, a culture,batch fermentation or continuous fermentation such that the dissolvedoxygen concentration in the medium remains between 0 and 10% ofsaturation. Substantially anaerobic conditions also includes growing orresting cells in liquid medium or on solid agar inside a sealed chambermaintained with an atmosphere of less than 1% oxygen. The percent ofoxygen can be maintained by, for example, sparging the culture with anN₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of methacrylic acid. Exemplary growthprocedures include, for example, fed-batch fermentation and batchseparation; fed-batch fermentation and continuous separation, orcontinuous fermentation and continuous separation. All of theseprocesses are well known in the art. Fermentation procedures areparticularly useful for the biosynthetic production of commercialquantities of methacrylic acid, 3-hydroxyisobutyric acid or2-hydroxyisobutyric acid. Generally, and as with non-continuous cultureprocedures, the continuous and/or near-continuous production ofmethacrylic acid, 3-hydroxyisobutyric acid or 2-hydroxyisobutyric acidwill include culturing a non-naturally occurring methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid producing organismof the invention in sufficient nutrients and medium to sustain and/ornearly sustain growth in an exponential phase. Continuous culture undersuch conditions can be include, for example, 1 day, 2, 3, 4, 5, 6 or 7days or more. Additionally, continuous culture can include 1 week, 2, 3,4 or 5 or more weeks and up to several months. Alternatively, organismsof the invention can be cultured for hours, if suitable for a particularapplication. It is to be understood that the continuous and/ornear-continuous culture conditions also can include all time intervalsin between these exemplary periods. It is further understood that thetime of culturing the microbial organism of the invention is for asufficient period of time to produce a sufficient amount of product fora desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of methacrylic acid, 3-hydroxyisobutyricacid or 2-hydroxyisobutyric acid can be utilized in, for example,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation. Examples of batch and continuous fermentation procedures arewell known in the art.

In addition to the above fermentation procedures using the methacrylicacid producers of the invention for continuous production of substantialquantities of methacrylic acid, the methacrylic acid producers also canbe, for example, simultaneously subjected to chemical synthesisprocedures to convert the product to other compounds or the product canbe separated from the fermentation culture and sequentially subjected tochemical conversion to convert the product to other compounds, ifdesired. Furthermore, in addition to the above fermentation proceduresusing the 3-hydroxyisobutyrate or 2-hydroxyisobutyrate producers of theinvention for continuous production of substantial quantities of3-hydroxyisobutyrate or 2-hydroxyisobutyrate, respectively, the3-hydroxyisobutyrate or 2-hydroxyisobutyrate producers also can be, forexample, simultaneously subjected to chemical synthesis procedures toconvert the product to other compounds or the product can be separatedfrom the fermentation culture and sequentially subjected to chemicalconversion to convert the product to other compounds, if desired.

One consideration for bioprocessing is whether to use a batch orcontinuous fermentation scheme. One difference between the two schemesthat will influence the amount of product produced is the presence of apreparation, lag, and stationary phase for the batch scheme in additionto the exponential growth phase. In contrast, continuous processes arekept in a state of constant exponential growth and, if properlyoperated, can run for many months at a time. For growth-associated andmixed-growth-associated product formation, continuous processes providemuch higher productivities (i.e., dilution rate times cell mass) due tothe elimination of the preparation, lag, and stationary phases.

Despite advantages in productivity, many more batch processes are inoperation than continuous processes for a number of reasons. First, fornon-growth associated product formation, the productivity of a batchsystem can significantly exceed that of a continuous process because thelatter would have to operate at very low dilution rates. Next,production strains generally have undergone modifications to theirgenetic material to improve their biochemical or protein productioncapabilities. These specialized strains are likely to grow less rapidlythan their parental complements whereas continuous processes such asthose employing chemostats (fermenters operated in continuous mode)impose large selection pressures for the fastest growing cells. Cellscontaining recombinant DNA or carrying point mutations leading to thedesired overproduction phenotype are susceptible to back-mutation intothe original less productive parental strain. It also is possible forstrains having single gene deletions to develop compensatory mutationsthat will tend to restore the wild-type growth phenotype. The fastergrowing cells usually out-compete their more productive counterparts forlimiting nutrients, drastically reducing productivity. Batch processes,on the other hand, limit the number of generations available by notreusing cells at the end of each cycle, thus decreasing the probabilityof the production strain reverting back to its wild-type phenotype.Finally, continuous processes are more difficult to operate long-termdue to potential engineering obstacles such as equipment failure andforeign organism contamination. The consequences of such failures alsoare much more considerable for a continuous process than with a batchculture.

For small-volume production of specialty chemicals and/or proteins, theproductivity increases of continuous processes rarely outweigh the risksassociated with strain stability and reliability. However, for theproduction of large-volume, growth-associated products such as3-hydroxyisobutyric acid or methacrylic acid, the increases inproductivity for a continuous process can result in significant economicgains when compared to a batch process. Although the engineeringobstacles associated with continuous bioprocess operation would alwaysbe present, the strain stability concerns can be overcome throughmetabolic engineering strategies that reroute metabolic pathways toreduce or avoid negative selective pressures and favor production of thetarget product during the exponential growth phase.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of methacrylic acid,3-hydroxyisobutyric acid or 2-hydroxyisobutyric acid.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework, Burgard et al., Biotechnol. Bioeng. 84:647-657(2003). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion strategies that result in genetically stablemicroorganisms which overproduce the target product. Specifically, theframework examines the complete metabolic and/or biochemical network ofa microorganism in order to suggest genetic manipulations that force thedesired biochemical to become an obligatory byproduct of cell growth. Bycoupling biochemical production with cell growth through strategicallyplaced gene deletions or other functional gene disruption, the growthselection pressures imposed on the engineered strains after long periodsof time in a bioreactor lead to improvements in performance as a resultof the compulsory growth-coupled biochemical production. Lastly, whengene deletions are constructed there is a negligible possibility of thedesigned strains reverting to their wild-type states because the genesselected by OptKnock are to be completely removed from the genome.Therefore, this computational methodology can be used to either identifyalternative pathways that lead to biosynthesis of a desired product orused in connection with the non-naturally occurring microbial organismsfor further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat enable an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.Analysis methods such as convex analysis, linear programming and thecalculation of extreme pathways as described, for example, in Schillinget al., J. Theor. Biol. 203:229-248 (2000); Schilling et al., Biotech.Bioeng. 71:286-306 (2000) and Schilling et al., Biotech. Prog.15:288-295 (1999), can be used to determine such phenotypiccapabilities.

As described above, one constraints-based method used in thecomputational programs applicable to the invention is flux balanceanalysis. Flux balance analysis is based on flux balancing in a steadystate condition and can be performed as described in, for example, Varmaand Palsson, Biotech. Bioeng. 12:994-998 (1994). Flux balance approacheshave been applied to reaction networks to simulate or predict systemicproperties of, for example, adipocyte metabolism as described in Felland Small, J. Biochem. 138:781-786 (1986), acetate secretion from E.coli under ATP maximization conditions as described in Majewski andDomach, Biotech. Bioeng. 35:732-738 (1990) or ethanol secretion by yeastas described in Vanrolleghem et al., Biotech. Prog. 12:434-448 (1996).Additionally, this approach can be used to predict or simulate thegrowth of S. cerevisiae on a variety of single-carbon sources as well asthe metabolism of H. influenzae as described in Edwards and Palsson,Proc. Natl. Acad. Sci. 97:5528-5533 (2000), Edwards and Palsson, J. Bio.Chem. 274:17410-17416 (1999) and Edwards et al., Nature Biotech.19:125-130 (2001).

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The ability of a cell or organism to obligatory couple growth to theproduction of a biochemical product can be illustrated in the context ofthe biochemical production limits of a typical metabolic networkcalculated using an in silico model. These limits are obtained by fixingthe uptake rate(s) of the limiting substrate(s) to their experimentallymeasured value(s) and calculating the maximum and minimum rates ofbiochemical production at each attainable level of growth. As shown inFIG. 16, the production of a desired biochemical generally is in directcompetition with biomass formation for intracellular resources. Underthese circumstances, enhanced rates of biochemical production willnecessarily result in sub-maximal growth rates. The knockouts suggestedby the metabolic modeling and simulation programs such as OptKnock, asdisclosed herein, are designed to restrict the allowable solutionboundaries forcing a change in metabolic behavior from the wild-typestrain as depicted in FIG. 16. Although the actual solution boundariesfor a given strain will expand or contract as the substrate uptakerate(s) increase or decrease, each experimental point will lie withinits calculated solution boundary. Plots such as these allow accuratepredictions of how close the designed strains are to their performancelimits which also indicates how much room is available for improvement.

The OptKnock mathematical framework is exemplified herein forpinpointing gene deletions leading to growth-coupled biochemicalproduction as illustrated in FIG. 16. The procedure builds uponconstraint-based metabolic modeling which narrows the range of possiblephenotypes that a cellular system can display through the successiveimposition of governing physico-chemical constraints (Price et al., NatRev Microbial, 2: 886-97 (2004)). As described above, constraint-basedmodels and simulations are well known in the art and generally invokethe optimization of a particular cellular objective, subject to networkstoichiometry, to suggest a likely flux distribution.

Briefly, the maximization of a cellular objective quantified as anaggregate reaction flux for a steady state metabolic network comprisinga set N={1, . . . , N} of metabolites and a set M={1, . . . , M} ofmetabolic reactions is expressed mathematically as follows:

$\begin{matrix}{maximize} & v_{{cellular}\mspace{14mu} {objective}} \\{{subject}\mspace{14mu} {to}} & {{{\sum\limits_{j = 1}^{M}{S_{ij}v_{j}}} = 0},{\forall{i \in N}}} \\\; & {v_{substrate} = {v_{substrate\_ uptake}\mspace{11mu} {mmol}\text{/}g\; D\; {W \cdot {hr}}}} \\{\forall{i \in \{ {{limiting}\mspace{14mu} {{substrate}(s)}} \}}} & \; \\\; & {v_{atp} \geq {v_{atp\_ main}{mmol}\text{/}g\; D\; {W \cdot {hr}}}} \\\; & {{v_{j} \geq 0},{\forall{j \in \{ {{irrev}.\mspace{11mu} {reactions}} \}}}}\end{matrix}\mspace{14mu}$

where S_(ij) is the stoichiometric coefficient of metabolite i inreaction j, ν_(j) is the flux of reaction j, ν_(substrate) _(—)_(uptake) represents the assumed or measured uptake rate(s) of thelimiting substrate(s), and ν_(atp) _(—) _(main) is the non-growthassociated ATP maintenance requirement. The vector ν includes bothinternal and external fluxes. In this study, the cellular objective isoften assumed to be a drain of biosynthetic precursors in the ratiosrequired for biomass formation, Neidhardt, F. C. et al., 2nd ed. 1996,Washington, D.C.: ASM Press. 2 v. (xx, 2822, 1xxvi). The fluxes aregenerally reported per 1 gDW·hr (gram of dry weight times hour) suchthat biomass formation is expressed as g biomass produced/gDW·hr or1/hr.

The modeling of gene deletions, and thus reaction elimination, firstemploys the incorporation of binary variables into the constraint-basedapproach framework, Burgard et al., Biotechnol Bioeng, 74: 364-375(2001), Burgard et al., Biotechnol Prog, 17: 791-797 (2001). Thesebinary variables,

$y_{j} = \{ {\begin{matrix}{1,} & {{if}\mspace{14mu} {reaction}\mspace{14mu} {flux}\mspace{14mu} v_{j}\mspace{14mu} {is}\mspace{14mu} {active}} \\{0,} & {{{if}\mspace{14mu} {reaction}\mspace{14mu} {flux}\mspace{14mu} v_{j}\mspace{14mu} {is}\mspace{14mu} {not}\mspace{14mu} {active}},}\end{matrix}{\forall{j \in \; M}}} $

assume a value of 1 if reaction j is active and a value of 0 if it isinactive. The following constraint,

ν_(j) ^(min) ·y _(j)≦ν_(j)≦ν_(j) ^(max) ·y _(j) ,∀jεM

ensures that reaction flux ν_(j) is set to zero only if variable y_(j)is equal to zero. Alternatively, when y_(j) is equal to one, ν_(j) isfree to assume any value between a lower ν_(j) ^(min) and an upper ν_(j)^(max) bound. Here, ν_(j) ^(min) and ν_(j) ^(max) are identified byminimizing and maximizing, respectively, every reaction flux subject tothe network constraints described above, Mahadevan et al., Metab Eng, 5:264-76 (2003).

Optimal gene/reaction knockouts are identified by solving a bileveloptimization problem that chooses the set of active reactions (y_(j)=1)such that an optimal growth solution for the resulting networkoverproduces the chemical of interest. Schematically, this bileveloptimization problem is illustrated in FIG. 2. Mathematically, thisbilevel optimization problem is expressed as the following bilevelmixed-integer optimization problem:

$\begin{matrix}{maximize} & v_{chemical} & \; & ({OptKnock}) & \; & \; & \; & \;\end{matrix}$ $\begin{pmatrix}{{subject}\mspace{14mu} {to}} & {maximize} & v_{biomass} & \; \\\; & {{subject}\mspace{14mu} {to}} & {{{\sum\limits_{j = 1}^{M}{S_{ij}v_{j}}} = 0},} & {\forall{i \in N}} \\\; & \; & {v_{substrate} = v_{substrate\_ uptake}} & \begin{matrix}{\forall{i \in}} \\\{ {{limiting}\mspace{14mu} {{substrate}(s)}} \}\end{matrix} \\\; & \; & {v_{atp} \geq v_{atp\_ main}} & \; \\\; & \; & {v_{biomass} \geq v_{biomass}^{target}} & \;\end{pmatrix}$ v_(j)^(min) ⋅ y_(j) ≤ v_(j) ≤ v_(j)^(max) ⋅ y_(j), ∀j ∈ M${\sum\limits_{j \in M^{forward}}^{\;}( {1 - y_{j}} )} = K$y_(j) ∈ {0, 1}, ∀j ∈ M

where ν_(chemical) is the production of the desired target product, forexample 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid, methacrylicacid, or other biochemical product, and K is the number of allowableknockouts. Note that setting K equal to zero returns the maximum biomasssolution of the complete network, while setting K equal to oneidentifies the single gene/reaction knockout (y_(j)=0) such that theresulting network involves the maximum overproduction given its maximumbiomass yield. The final constraint ensures that the resulting networkmeets a minimum biomass yield. Burgard et al., Biotechnol Bioeng, 84:647-57 (2003), provide a more detailed description of the modelformulation and solution procedure. Problems containing hundreds ofbinary variables can be solved in the order of minutes to hours usingCPLEX 8.0, GAMS: The Solver Manuals. 2003: GAMS Development Corporation,accessed via the GAMS, Brooke et al., GAMS Development Corporation(1998), modeling environment on an IBM RS6000-270 workstation. TheOptKnock framework has already been able to identify promising genedeletion strategies for biochemical overproduction, Burgard et al.,Biotechnol Bioeng, 84: 647-57 (2003), Pharkya et al., Biotechnol Bioeng,84: 887-899 (2003), and establishes a systematic framework that willnaturally encompass future improvements in metabolic and regulatorymodeling frameworks.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

Constraints of the above form preclude identification of larger reactionsets that include previously identified sets. For example, employing theinteger cut optimization method above in a further iteration wouldpreclude identifying a quadruple reaction set that specified reactions1, 2, and 3 for disruption since these reactions had been previouslyidentified. To ensure identification of all possible reaction setsleading to growth-coupled production of a product, a modification of theinteger cut method was employed.

Briefly, the modified integer cut procedure begins with iteration ‘zero’which calculates the maximum production of the desired biochemical atoptimal growth for a wild-type network. This calculation corresponds toan OptKnock solution with K equaling 0. Next, single knockouts areconsidered and the two parameter sets, objstore_(iter) andystore_(iter,j) are introduced to store the objective function(ν_(chemical)) and reaction on-off information (y_(j)), respectively, ateach iteration, iter. The following constraints are then successivelyadded to the OptKnock formulation at each iteration.

ν_(chemical)≧objstore_(iter) +ε−M·Σ _(jεystore) _(iter,j) ₌₀ y _(j)

In the above equation, ε and M are a small and a large numbers,respectively. In general, ε can be set at about 0.01 and M can be set atabout 1000. However, numbers smaller and/or larger then these numbersalso can be used. M ensures that the constraint can be binding only forpreviously identified knockout strategies, while ε ensures that addingknockouts to a previously identified strategy must lead to an increaseof at least ε in biochemical production at optimal growth. The approachmoves onto double deletions whenever a single deletion strategy fails toimprove upon the wild-type strain. Triple deletions are then consideredwhen no double deletion strategy improves upon the wild-type strain, andso on. The end result is a ranked list, represented as desiredbiochemical production at optimal growth, of distinct deletionstrategies that differ from each other by at least one knockout. Thisoptimization procedure as well as the identification of a wide varietyof reaction sets that, when disrupted, lead to the growth-coupledproduction of a biochemical product are exemplified in detail furtherbelow. Given the teachings and guidance provided herein, those skilledin the art will understand that the methods and metabolic engineeringdesigns exemplified herein are applicable to the obligatory coupling ofcell or microorganism growth to any biochemical product.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, an OptKnock strategy was used to identify geneknockouts to couple growth with production of a desired product such as3-hydroxyisobutyric acid or methacrylic acid (see Examples XXI to XXIII)While identified using an OptKnock strategy, it is understood that anysuitable modeling system, including a system such as SimPheny™ can beused to identity gene knockouts to develop strains able to coupleproduction of a desired product to growth, as disclosed herein. Any ofthe gene deletion strategies disclosed herein can be combined, asappropriate, with any of the non-naturally occurring microbial organismsdisclosed herein having a pathway for production of 2-hydroxyisobutyricacid, 3-hydroxyisobutyric acid or methacrylic acid to increaseproduction of 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid ormethacrylic acid.

Thus the invention additionally provides a non-naturally occurringmicrobial organism, comprising one or more gene disruptions, the one ormore gene disruptions occurring in genes encoding proteins or enzymeswhere the one or more gene disruptions confer increased production of3-hydroxyisobutyric acid or methacrylic acid in said organism. Asdisclosed herein, the gene disruptions can confer production of3-hydroxyisobutyric acid or methacrylic acid that is growth-coupled ornot growth-coupled. For example, the one or more gene disruptions canencode a protein or enzyme listed in Tables 10 or 11 (see Examples XXIIand XXIII). In a particular embodiment, the one or more gene disruptionscan encode proteins or enzymes selected from the group consisting ofmalate dehydrogenase, lactate dehydrogenase and acetaldehyde-CoAdehydrogenase. In an additional embodiment, the organism can furthercomprise one or more gene disruptions encoding proteins or enzymesselected from the group consisting of aspartase, pyruvate formate lyase,NAD(P) transhydrogenase, glutamate dehydrogenase, ATP synthase,phosphoenolpyruvate:pyruvate phosphotransferase system, glutamatedehydrogenase, phosphotransacetylase, acetate kinase,6-phosphogluconolactonase, glucose 6-phosphate dehydrogenase and NADHdehydrogenase.

Thus, the invention provides an organism with an improved yield of MAAor 3-HIB (see Examples XXII and XXIII) that contains functionaldisruptions in alcohol dehydrogenase, malate dehydrogenase, and lactatedehydrogenase (Tables 6 and 8, Design 1). Additionally provided is anorganism with an additional functional disruption in any of glutamatedehydrogenase, aspartase, NAD(P) transhydrogenase or NADH dehydrogenase(Table 6, Designs 2, 7, 10, 13; Table 8, Designs 2, 8). Further providedis an organism with an additional functional disruption in aspartase andany of NAD(P) transhydrogenase, glutamate dehydrogenase, ATP synthase orpyruvate formate lyase (Table 6, Designs 3, 5; Table 8, Designs 3, 5).Also provided is an organism with an additional functional disruption inpyruvate formate lyase and any of NAD(P) transhydrogenase or glutamatedehydrogenase (Table 6, Design 4; Table 8, Design 4). Additionallyprovided is an organism with an additional functional disruption in ATPsynthase and in any of pyruvate formate lyase, D-glucose transport viaPEP:Pyr PTS, 6-phosphogluconolactonase or glucose-6-phosphatedehydrogenase (Table 6, Design 6; Table 8, Design 6, 7). Also providedis an organism with an additional functional disruption in glutamatedehydrogenase and pyruvate formate lyase (Table 6 Design 8). Furtherprovided is an organism with an additional functional disruption in anyof acetate kinase or phosphotransacetylase (Table 6, Design 9).Additionally provided is an organism with an additional functionaldisruption in NAD(P) transhydrogenase and in any of6-phosphogluconolactonase or glucose-6-phosphate dehydrogenase (Table 6,Design 11; Table 8, Design 9 w/THD2). Further provided is an organismwith an additional functional disruption in glutamate dehydrogenase andin any of 6-phosphogluconolactonase or glucose-6-phosphate dehydrogenase(Table 8, Design 9 w/GLUDy). Also provided is an organism with anadditional functional disruption in pyruvate formate lyase (Table 6,Design 12). Additionally provided is an organism with an additionalfunctional disruption in NADH dehydrogenase and in any of acetate kinaseor phosphotransacetylase (Table 6, Design 14).

As disclosed herein, the one or more gene disruptions can comprises adeletion of the one or more genes. Such methods for gene disruptions,including gene deletions, are well known to those skilled in the art, asdisclosed herein. If desired, the cells can be cultured in asubstantially anaerobic culture medium.

Also provided are methods for producing 3-hydroxyisobutyric acid ormethacrylic acid using the organisms disclosed herein and discussedabove and in Examples XXII and XXIII having one or more genedisruptions. Thus, the invention provides a method for producing3-hydroxyisobutyric acid or methacrylic acid comprising culturing anon-naturally occurring microbial organism, comprising one or more genedisruptions, the one or more gene disruptions occurring in genesencoding a protein or enzyme wherein the one or more gene disruptionsconfer obligatory coupling of 3-hydroxyisobutyric acid or methacrylicacid production to growth of the organism when the gene disruptionreduces an activity of the protein or enzyme, whereby said one or moregene disruptions confers stable growth-coupled production of3-hydroxyisobutyric acid or methacrylic acid onto the organism.

Given the teachings and guidance provided herein, those skilled in theart will understand that to disrupt an enzymatic reaction it isnecessary to disrupt the catalytic activity of the one or more enzymesinvolved in the reaction. Disruption can occur by a variety of meansincluding, for example, deletion of an encoding gene or incorporation ofa genetic alteration in one or more of the encoding gene sequences. Theencoding genes targeted for disruption can be one, some, or all of thegenes encoding enzymes involved in the catalytic activity. For example,where a single enzyme is involved in a targeted catalytic activitydisruption can occur by a genetic alteration that reduces or destroysthe catalytic activity of the encoded gene product. Similarly, where thesingle enzyme is multimeric, including heteromeric, disruption can occurby a genetic alteration that reduces or destroys the function of one orall subunits of the encoded gene products. Destruction of activity canbe accomplished by loss of the binding activity of one or more subunitsin order to form an active complex, by destruction of the catalyticsubunit of the multimeric complex or by both. Other functions ofmultimeric protein association and activity also can be targeted inorder to disrupt a metabolic reaction of the invention. Such otherfunctions are well known to those skilled in the art. Further, some orall of the functions of a single polypeptide or multimeric complex canbe disrupted according to the invention in order to reduce or abolishthe catalytic activity of one or more enzymes involved in a reaction ormetabolic modification of the invention. Similarly, some or all ofenzymes involved in a reaction or metabolic modification of theinvention can be disrupted so long as the targeted reaction isdestroyed.

Given the teachings and guidance provided herein, those skilled in theart also will understand that an enzymatic reaction can be disrupted byreducing or eliminating reactions encoded by a common gene and/or by oneor more orthologs of that gene exhibiting similar or substantially thesame activity. Reduction of both the common gene and all orthologs canlead to complete abolishment of any catalytic activity of a targetedreaction. However, disruption of either the common gene or one or moreorthologs can lead to a reduction in the catalytic activity of thetargeted reaction sufficient to promote coupling of growth to productbiosynthesis. Exemplified herein are both the common genes encodingcatalytic activities for a variety of metabolic modifications as well astheir orthologs. Those skilled in the art will understand thatdisruption of some or all of the genes encoding a enzyme of a targetedmetabolic reaction can be practiced in the methods of the invention andincorporated into the non-naturally occurring microbial organisms of theinvention in order to achieve the growth-coupled product production.

In some embodiments, the gene disruption can include a complete genedeletion. In some embodiments other means to disrupt a gene include, forexample, frameshifting by omission or addition of oligonucleotides or bymutations that render the gene inoperable. One skilled in the art willrecognize the advantages of gene deletions, however, because of thestability it may confer to the non-naturally occurring organism fromreverting to its wild-type. In particular, the gene disruptions areselected from the gene set that includes genes detailed herein.

Each of the proposed strains can be supplemented with additionaldeletions if it is determined that the predicted strain designs do notsufficiently couple the formation of the product with biomass formation.Alternatively, some other enzymes not known to possess significantactivity under the growth conditions can become active due to adaptiveevolution or random mutagenesis and can also be knocked out. However,the list of gene deletion sets provided here serves as a starting pointfor construction of high-yielding growth-coupled 3-hydroxyisobutyricacid or methacrylic acid production strains.

One skilled in the art will recognize the ability to also produce MAA,2-hydroxyisobutyrate, or 3-hydroxyisobutyrate, by non-growth-coupledproduction by providing a non-producing growth phase, followed by anon-growth production phase, for example. The results described hereinindicate that combinations of gene deletions or functional disruptionsof genes significantly improve the MAA, 2-hydroxyisobutyrate, or3-hydroxyisobutyrate production capabilities of E. coli and otherorganisms. The strain design pathways are equally applicable if amicrobial organism other than E. coli is chosen as the production host,even if the organism naturally lacks the activity or exhibits lowactivity of a subset of the gene products marked for disruption. In thelatter case, disruptions can be introduced to eliminate or lessen theenzymatic activities of the gene products that are naturally present inthe chosen production host. In some embodiments, growth-coupledproduction of MAA, 2-hydroxyisobutyrate, or 3-hydroxyisobutyrate for thein silico determined metabolic pathways is confirmed by construction ofstrains having the designed metabolic genotype. These metabolicallyengineered cells or organisms can also be subjected to adaptiveevolution to further augment growth-coupled product production. In someembodiments, the engineered cells or organisms can also incorporateadditional copies of beneficial genes to increase flux through aparticular metabolic pathway. Alternatively, exogenous gene insertionsfrom another organism can be used to install functionality that is notpresent in the host organism.

The design strategies described herein are useful not only for enhancinggrowth coupled production, but they are also well-suited for enhancingnon-growth coupled production because they link the production of2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acidto energy generation and/or redox balance. Exemplary non-growth coupledproduction methods include implementing an aerobic growth phase followedby an anaerobic production phase. For example, Vemuri et al., (J. Ind.Microbiol. Biotechnol. 28 (6):325-332 (2002)) describe a dual-phaseprocess for the production of succinate in E. Coli. Okino et al. Appl.Microbiol. Biotechnol. 81 (3):459-464 (2008)) describe a similarnon-growth coupled production process in a strain of Corynebacteriumglutamicum strain.

Another such method involves withholding an essential nutrient from apropogated cell culture, thereby limiting growth, but not precludingproduction as described in Durner et al., Appl. Environ. Microbiol. 66(8):3408-3414 (2000). Yet another strategy aimed at decoupling growthfrom production involves replacing the growth substrate with anothercompound that is more slowly metabolizable as described in Altamirano etal., Biotechnol. Bioeng. 76:351-360 (2001). Growth decoupled-productformation can also be brought about by specific genetic modifications asdescribed in Blombach et al., Appl. Microbiol. Biotechnol. 79:471-479(2008).

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example 1 Pathway for Conversion of Succinyl-CoA to MAA via3-Hydroxyisobutyrate

This example describes an exemplary MAA synthesis pathway fromsuccinyl-CoA to methacrylic acid via 3-hydroxyisobutyrate.

One exemplary pathway for MAA synthesis proceeds from succinyl-CoA (seeFIG. 2). This pathway uses at least three and at most five enzymaticsteps to form MAA from succinyl-CoA. The pathway is redox-balanced,indicating that it can potentially lead to the maximum MAA yield of 1.33mol per mol of glucose under anaerobic conditions with no byproductformation. Moreover, the pathway is energetically efficient and cangenerate 0.5 ATP per mole of glucose metabolized to MAA ifphosphoenolpyruvate (PEP) carboxykinase (PEPCK) activity is assumedirreversible (that is, cannot catalyze the ATP-generating carboxylationof PEP to oxaloacetate) or 1.72 ATP if PEPCK is assumed reversible. Thelatter ATP yield is comparable to the ATP yield from the formation oflactate or ethanol from glucose, that is, 2 ATP per glucose metabolized,indicating that methacrylate fermentation can generate sufficient energyfor cell growth and maintenance.

In this pathway (see FIG. 2), succinyl-CoA is first converted to(R)-methylmalonyl-CoA, which is potentially converted to(S)-methylmalonyl-CoA by an epimerase. Either the (R)- or(S)-stereoisomer of methylmalonyl-CoA is then reduced to (R)- or(S)-3-hydroxyisobutyrate, respectively, by either a pair of enzymes (asshown in FIG. 2) or a single enzyme that exhibits acyl-CoA reductase andalcohol dehydrogenase activities. The pathway from succinyl-CoA to3-hydroxyisobutyrate has also been described in WO 2007/141208. In thefinal step, 3-hydroxyisobutyrate is dehydrated to form MAA.

Successfully engineering this pathway involves identifying anappropriate set of enzymes with sufficient activity and specificity.This entails identifying an appropriate set of enzymes, cloning theircorresponding genes into a production host, optimizing fermentationconditions, and assaying for product formation following fermentation.To engineer a production host for the production of methacrylic acid,one or more exogenous DNA sequence(s) are expressed in microorganisms.In addition, the microorganisms can have endogenous gene(s) functionallydeleted. These modifications allow the production of methacrylic acidusing renewable feedstock.

Below is described a number of biochemically characterized candidategenes capable of encoding enzymes that catalyze each step of the desiredpathway. Although described using E. coli as a host organism to engineerthe pathway, essentially any suitable host organism can be used.Specifically listed are genes that are native to E. coli as well asgenes in other organisms that can be applied to catalyze the appropriatetransformations when properly cloned and expressed.

Referring to FIG. 2, step 1 involves methylmalonyl-CoA mutase (EC5.4.99.2). In the first step, succinyl-CoA is converted intomethylmalonyl-CoA by methylmalonyl-CoA mutase (MCM). In E. coli, thereversible adenosylcobalamin-dependant mutase participates in athree-step pathway leading to the conversion of succinate to propionate(Haller et al., Biochemistry 39:4622-4629 (2000)). Overexpression of theMCM gene candidate along with the deletion of YgfG can be used toprevent the decarboxylation of methylmalonyl-CoA to propionyl-CoA and tomaximize the methylmalonyl-CoA available for MAA synthesis. MCM isencoded by genes scpA in Escherichia coli (Bobik and Rasche, Anal.Bioanal. Chem. 375:344-349 (2003); Haller et al., Biochemistry39:4622-4629 (2000)) and mutA in Homo sapiens (Padovani and Banerjee,Biochemistry 45:9300-9306 (2006)). In several other organisms MCMcontains alpha and beta subunits and is encoded by two genes. Exemplarygene candidates encoding the two-subunit protein are Propionibacteriumfredenreichii sp. shermani mutA and mutB (Korotkova and Lidstrom, J.Biol. Chem. 279:13652-13658 (2004)) and Methylobacterium extorquens mcmAand mcmB (Korotkova and Lidstrom, supra, 2004). The protein sequences ofthese genes can be identified by their corresponding GenBank accessionnumbers.

Gene GenBank ID Organism scpA NP_417392.1 Escherichia coli K12 mutAP22033.3 Homo sapiens mutA P11652.3 Propionibacterium fredenreichii sp.shermanii mutB P11653.3 Propionibacterium fredenreichii sp. shermaniimcmA Q84FZ1 Methylobacterium extorquens mcmB Q6TMA2 Methylobacteriumextorquens

These sequences can be used to identify homologue proteins in GenBank orother databases through sequence similarity searches (for example,BLASTp). The resulting homologue proteins and their corresponding genesequences provide additional exogenous DNA sequences for transformationinto E. coli or other suitable host microorganisms to generateproduction hosts. Additional gene candidates include the following,which were identified based on high homology to the E. coli spcA geneproduct.

Gene GenBank ID Organism sbm NP_838397.1 Shigella flexneri SARI_04585ABX24358.1 Salmonella enterica YfreA_01000861 ZP_00830776.1 Yersiniafrederiksenii

There exists evidence that genes adjacent to the methylmalonyl-CoAmutase catalytic genes contribute to maximum activity. For example, ithas been demonstrated that the meaB gene from M. extorquens forms acomplex with methylmalonyl-CoA mutase, stimulates in vitro mutaseactivity, and possibly protects it from irreversible inactivation(Korotkova and Lidstrom, J. Biol. Chem. 279:13652-13658 (2004)). The M.extorquens meaB gene product is highly similar to the product of the E.coli argK gene (BLASTp: 45% identity, e-value: 4e-67), which is adjacentto scpA on the chromosome. No sequence for a meaB homolog in P.freudenreichii is catalogued in GenBank. However, the Propionibacteriumacnes KPA171202 gene product, YP_(—)055310.1, is 51% identical to the M.extorquens meaB protein and its gene is also adjacent to themethylmalonyl-CoA mutase gene on the chromosome.

Gene GenBank ID Organism argK AAC75955.1 Escherichia coli K12YP_055310.1 Propionibacterium acnes KPA171202 meaB 2QM8_BMethylobacterium extorquens

E. coli can synthesize adenosylcobalamin, a necessary cofactor for thisreaction, only when supplied with the intermediates cobinamide orcobalamin (Lawrence and Roth. J. Bacteriol. 177:6371-6380 (1995);Lawrence and Roth, Genetics 142:11-24 (1996)). Alternatively, theability to synthesize cobalamins de novo has been conferred upon E. colifollowing the expression of heterologous genes (Raux et al., J.Bacteriol. 178:753-767 (1996)).

Referring to FIG. 2, step 2 involves methylmalonyl-CoA epimerase (EC5.1.99.1). The second enzyme in the pathway, methylmalonyl-CoA epimerase(MMCE), converts (R)-methylmalonyl-CoA to (S)-methylmalonyl-CoA. MMCE isan essential enzyme in the breakdown of odd-numbered fatty acids and ofthe amino acids valine, isoleucine, and methionine. Methylmalonyl-CoAepimerase activity is not believed to be encoded in the E. coli genome(Boynton et al., J. Bacteriol. 178:3015-3024 (1996)), but is present inother organisms such as Homo sapiens (YqjC) (Fuller and Leadlay,Biochem. J. 213:643-650 (1983)), Rattus norvegicus (Mcee) (Bobik andRasche, J. Biol. Chem. 276:37194-37198 (2001)), Propionibacteriumshermanii (AF454511) (Fuller. and Leadlay, Biochem. J. 213:643 -650(1983); Haller et al., Biochemistry 39:4622-4629 (2000); McCarthy etal., Structure 9:637-646.2001)) and Caenorhabditis elegans (mmce) (Kuhnlet al., FEBS J. 272:1465-1477 (2005)). This enzymatic step may or maynot be necessary depending upon the stereospecificity of the enzyme orenzymes used for the conversion of methylmalonyl-CoA to3-hydroxyisobutyrate (steps 3-4 in FIG. 2). Additional gene candidatesin microorganisms, such as AE016877 in Bacillus cereus, have highsequence homology but have not been experimentally verified.

Gene GenBank ID Organism MCEE Q96PE7.1 Homo sapiens Mcee_predictedNP_001099811.1 Rattus norvegicus AF454511 AAL57846.1 Propionibacteriumfredenreichii sp. shermanii mmce AAT92095.1 Caenorhabditis elegansAE016877 AAP08811.1 Bacillus cereus ATCC 14579

Referring to FIG. 2, step 3 involves methylmalonyl-CoA reductase (EC1.2.1.-). As shown in FIG. 2, the reduction of methylmalonyl-CoA to itscorresponding alcohol, 3-hydroxyisobutyrate, can proceed by twoenzymatic steps. The first step, conversion of methylmalonyl-CoA tomethylmalonic semialdehyde, is accomplished by a CoA-dependent aldehydedehydrogenase. An enzyme encoded by a malonyl-CoA reductase gene fromSulfolobus tokodaii (Alber et. al., J. Bacteriol. 188(24):8551-8559(2006)), has been shown to catalyze the conversion of methylmalonyl-CoAto its corresponding aldehyde (WO2007141208). A similar enzyme exists inMetallosphaera sedula (Alber et. al., J. Bacteriol. 188 (24):8551-8559(2006)). Several additional CoA dehydrogenases are capable also ofreducing an acyl-CoA to its corresponding aldehyde. Exemplary genes thatencode such enzymes include the Acinetobacter calcoaceticus acr1encoding a fatty acyl-CoA reductase (Reiser and Somerville. J.Bacteriol. 179:2969-2975 (1997)), the Acinetobacter sp. M-1 fattyacyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol.68:1192-1195 (2002)), and a CoA- and NADP-dependent succinatesemialdehyde dehydrogenase encoded by the sucD gene in Clostridiumkluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996);Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). The enzymeacylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG,is also a good candidate as it has been demonstrated to oxidize andacylate the branched-chain compound isobutyraldehyde (Powlowski et al.,J. Bacteriol. 175:377-385 (1993)).

Gene GenBank ID Organism mcr NP_378167 Sulfolobus tokodaii mcrYP_001190808.1 Metallosphaera sedula acr1 YP_047869.1 Acinetobactercalcoaceticus acr1 AAC45217 Acinetobacter baylyi acr1 BAB85476.1Acinetobacter sp. Strain M-1 sucD P38947.1 Clostridium kluyveri bphGBAA03892.1 Pseudomonas sp

Referring to FIG. 2, step 4 involves 3-hydroxyisobutyrate dehydrogenase(EC 1.1.1.31). 3-hydroxyisobutyrate dehydrogenase catalyzes thereversible oxidation of 3-hydroxyisobutyrate to methylmalonatesemialdehyde. This enzyme participates in valine, leucine and isoleucinedegradation and has been identified in bacteria, eukaryotes, andmammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 hasbeen structurally characterized (Lokanath et al., J. Mol. Biol.352:905-917 (2005)). The reversibility of the human 3-hydroxyisobutyratedehydrogenase was demonstrated using isotopically-labeled substrate(Manning and Pollitt, Biochem. J. 231:481-484 (1985)). Additional genesencoding this enzyme include 3hidh in Homo sapiens (Hawes et al.,Methods Enzymol. 324:218-228 (2000)) and Oryctolagus cuniculus(Chowdhury et al., Biosci. Biotechnol. Biochem. 60:2043-2047 (1996);Hawes et al., Methods Enzymol. 324:218-228 (2000)), mmsb in Pseudomonasaeruginosa, and dhat in Pseudomonas putida (Aberhart and Hsu. J Chem.Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al., Biosci.Biotechnol. Biochem. 67:438-441 (2003); Chowdhury et al., Biosci.Biotechnol. Biochem. 60:2043-2047 (1996)).

Gene GenBank ID Organism P84067 P84067 Thermus thermophilus mmsbP28811.1 Pseudomonas aeruginosa dhat Q59477.1 Pseudomonas putida 3hidhP31937.2 Homo sapiens 3hidh P32185.1 Oryctolagus cuniculus

Referring to FIG. 2, as an alternative, steps 3 and 4 can involve acombined Alcohol/Aldehyde dehydrogenase (EC 1.2.1.-). Methylmalonyl-CoAcan be reduced to 3-hydroxyisobutyrate in one step by a multifunctionalenzyme with dual acyl-CoA reductase and alcohol dehydrogenase activity.No evidence for the direct conversion of methylmalonyl-CoA to3-hydroxyisobutyrate has been reported. However, this reaction issimilar to the common conversions such as acetyl-CoA to ethanol andbutyryl-CoA to butanol, which are catalyzed by CoA-dependant enzymeswith both alcohol and aldehyde dehydrogenase activities. Gene candidatesinclude the E. coli adhE (Kessler et al., FEBS Lett. 281:59-63 (1991))and C. acetobutylicum bdh I and bdh II (Walter, et al., J. Bacteriol.174:7149-7158 (1992)), which can reduce acetyl-CoA and butyryl-CoA toethanol and butanol, respectively. In addition to reducing acetyl-CoA toethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides hasbeen shown to oxide the branched chain compound isobutyraldehyde toisobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55(1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)). An additionalcandidate enzyme for converting methylmalonyl-CoA directly to3-hydroxyisobutyrate is encoded by a malonyl-CoA reductase fromChloroflexus aurantiacus (Hügler, et al., J. Bacteriol. 184(9):2404-2410 (2002).

Gene GenBank ID Organism mcr YP_001636209.1 Chloroflexus aurantiacusadhE NP_415757.1 Escherichia coli bdh I NP_349892.1 Clostridiumacetobutylicum bdh II NP_349891.1 Clostridium acetobutylicum adhEAAV66076.1 Leuconostoc mesenteroides

Referring to FIG. 2, step 5 involves 3-hydroxyisobutyrate dehydratase(EC 4.2.1.-). The final step involves the dehydration of3-hydroxyisobutyrate to methacrylic acid. No direct evidence for thisspecific enzymatic transformation has been identified. However, mostdehydratases catalyze the α,β-elimination of water, which involvesactivation of the α-hydrogen by an electron-withdrawing carbonyl,carboxylate, or CoA-thiol ester group and removal of the hydroxyl groupfrom the β-position (Buckel and Barker, J Bacteriol. 117:1248-1260(1974); Martins et al, Proc. Natl. Acad. Sci. USA 101:15645-15649(2004)). This is the exact type of transformation proposed for the finalstep in the methacrylate pathway. In addition, the proposedtransformation is highly similar to the 2-(hydroxymethyl)glutaratedehydratase of Eubacterium barkeri (FIG. 3A). This enzyme has beenstudied in the context of nicotinate catabolism and is encoded by hmd(Alhapel et al., Proc. Natl. Acad. Sci. USA 103:12341-12346 (2006)).Similar enzymes with high sequence homology are found in Bacteroidescapillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius.These enzymes are also homologous to the α- and β-subunits of[4Fe-4S]-containing bacterial serine dehydratases, for example, E. colienzymes encoded by tdcG, sdhB, and sdaA).

Gene GenBank ID Organism hmd ABC88407.1 Eubacterium barkeri BACCAP_02294ZP_02036683.1 Bacteroides capillosus ATCC 29799 ANACOL_02527ZP_02443222.1 Anaerotruncus colihominis DSM 17241 NtherDRAFT_2368ZP_02852366.1 Natranaerobius thermophilus JW/NM-WN-LF

Fumarate hydratase enzymes, which naturally catalyze the dehydration ofmalate to fumarate, represent an additional set of candidates (FIG. 3B).Although the ability of fumarate hydratase to react on branchedsubstrates has not been described, a wealth of structural information isavailable for this enzyme and other researchers have successfullyengineered the enzyme to alter activity, inhibition and localization(Weaver, Acta Crystallogr. D Biol. Crystallogr. 61:1395-1401 (2005)).Exemplary enzyme candidates include those encoded by fumC fromEscherichia coli (Estevez et al., Protein Sci. 11:1552-1557 (2002); Hongand Lee, Biotechnol. Bioprocess Eng. 9:252-255 (2004); Rose and Weaver,Proc. Natl. Acad. Sci. USA 101:3393-3397 (2004)), Campylobacter jejuni(Smith et al., Int. J. Biochem. Cell Biol. 31:961-975 (1999)) andThermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55(1998)), and fumH from Rattus norvegicus (Kobayashi et al., J. Biochem.89:1923-1931 (1981)). Similar enzymes with high sequence homologyinclude fum1 from Arabidopsis thaliana and fumC from Corynebacteriumglutamicum.

Gene name GenBankID Organism fumC P05042.1 Escherichia coli K12 fumCO69294.1 Campylobacter jejuni fumC P84127 Thermus thermophilus fumHP14408.1 Rattus norvegicus fum1 P93033.2 Arabidopsis thaliana fumCQ8NRN8.1 Corynebacterium glutamicum

This example describes a biosynthetic pathway for production of MMA fromsuccinyl-CoA.

Example II Preparation of an MAA Producing Microbial Organism Having aPathway for Converting Succinyl-CoA to MAA via 3-Hydroxyisobutyrate

This example describes the generation of a microbial organism capable ofproducing MAA from succinyl-CoA via 3-hydroxyisobutyrate.

Escherichia coli is used as a target organism to engineer the MAApathway shown in FIG. 2. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing MAA. E. coliis amenable to genetic manipulation and is known to be capable ofproducing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions.

To generate an E. coli strain engineered to produce MAA, nucleic acidsencoding the enzymes utilized in the pathway are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the scpA (NP417392.1), argK (AAC75955.1), and AF454511 (AAL57846.1) genes encodingthe methylmalonyl-CoA mutase, its stabilizer protein, andmethylmalonyl-CoA epimerase activities, respectively, are cloned intothe pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. In addition, mcr (NP_(—)378167), dhat (Q59477.1), and hmd(ABC88407.1) genes encoding methylmalonyl-CoA reductase,3-hydroxyisobutyrate dehydrogenase, and 3-hydroxyisobutyrate dehydrataseactivities, respectively, are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. The two sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for MAA synthesis via the succinyl-CoA to3-hydroxyisobutyrate pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the MAA synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce MAAis confirmed using HPLC, gas chromatography-mass spectrometry (GCMS)and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MAA synthesis pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of MAA. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of MAA. Adaptiveevolution also can be used to generate better producers of, for example,the succinyl-CoA intermediate of the MAA product. Adaptive evolution isperformed to improve both growth and production characteristics (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science314:1565-1568 (2006)). Based on the results, subsequent rounds ofmodeling, genetic engineering and adaptive evolution can be applied tothe MAA producer to further increase production.

For large-scale production of MAA, the above organism is cultured in afermenter using a medium known in the art to support growth of theorganism under anaerobic conditions. Fermentations are performed ineither a batch, fed-batch or continuous manner Anaerobic conditions aremaintained by first sparging the medium with nitrogen and then sealingthe culture vessel, for example, flasks can be sealed with a septum andcrimp-cap. Microaerobic conditions also can be utilized by providing asmall hole in the septum for limited aeration. The pH of the medium ismaintained at a pH of around 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce MAA from succinyl-CoA via 3-hydroxyisobutyrate.

Example III Pathway for Conversion of Succinyl-CoA to MAA via3-Amino-2-Methylpropanoate

This example describes an exemplary MAA synthesis pathway fromsuccinyl-CoA to MAA via 3-amino-methylpropanoate.

Another exemplary pathway for MAA biosynthesis proceeds fromsuccinyl-CoA through 3-amino-2-methylpropanoate (see FIG. 4). Thispathway is high-yielding under anaerobic conditions with a maximumtheoretical yield of 1.33 mol MAA/mol glucose. The pathway is alsoenergetically efficient, capable of generating 1.55 mol ATP/mol glucoseat maximum product yield, under the assumption that PEP carboxykinasecan operate reversibly.

The first three steps of this pathway, involving the conversion ofsuccinyl-CoA to methylmalonate semialdehyde, are identical to thesuccinyl-CoA to MAA pathway described in Example I (see FIG. 2). Thepathway diverges at step 4, where methylmalonate semialdehyde isconverted to 3-amino-2-methylpropionate by a transaminase The finalpathway step entails deamination of 3-amino-2-methylpropionate tomethacrylic acid.

Enzyme and gene candidates for catalyzing the first three pathway stepsare described in Example I. Gene candidates for steps 4 and 5 arediscussed below.

Referring to FIG. 4, step 4 involves 3-amino-2-methylpropionatetransaminase (EC 2.6.1.22). 3-amino-2-methylpropionate transaminasecatalyzes the transformation from methylmalonate semialdehyde to3-amino-2-methylpropionate. The enzyme, characterized in Rattusnorvegicus and Sus scrofa and encoded by Abat, has been shown tocatalyze this transformation in the direction of interest in the pathway(Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); Tamaki etal., Methods Enzymol. 324:376-389 (2000)). Enzyme candidates in otherorganisms with high sequence homology to 3-amino-2-methylpropionatetransaminase include Gta-1 in C. elegans and gabT in Bacillus subtilus.Additionally, one of the native GABA aminotransferases in E. coli,encoded by gene gabT, has been shown to have broad substrate specificityand may utilize 3-amino-2-methylpropionate as a substrate (Liu et al.,Biochemistry 43:10896-10905 (2004); Schulz et al., Appl. Environ.Microbiol. 56:1-6 (1990)).

Gene name GenBankID Organism Abat P50554.3 Rattus norvegicus AbatP80147.2 Sus scrofa Gta-1 Q21217.1 Caenorhabditis elegans gabT P94427.1Bacillus subtilus gabT P22256.1 Escherichia coli K12

Referring to FIG. 4, step 5 involves 3-amino-2-methylpropionate ammonialyase (EC 4.3.1.-). In the final step of this pathway,3-amino-2-methylpropionate is deaminated to methacrylic acid. An enzymecatalyzing this exact transformation has not been demonstratedexperimentally; however the native E. coli enzyme, aspartate ammonialyase (EC 4.3.1.1), may be able to catalyze this reaction (see FIG. 5A).Encoded by aspA in E. coli, aspartate ammonia lyase deaminatesasparatate to form fumarate but can also react with alternate substratesaspartatephenylmethylester, asparagine, benzyl-aspartate and malate (Maet al., Ann. N.Y. Acad. Sci. 672:60-65 (1992)). In a separate study,directed evolution was been employed on this enzyme to alter substratespecificity (Asano et al., Biomol. Eng. 22:95-101 (2005)). Genesencoding aspartase in other organisms include ansB in Bacillus subtilus(Sjostrom et al., Biochim. Biophys. Acta 1324:182-190 (1997)) and aspAin Pseudomonas fluorescens (Takagi et al., J. Biochem. 96:545-552(1984); Takagi et al., J. Biochem. 100:697-705 (1986)) and Serratiamarcescens (Takagi et al., J. Bacteriol. 161:1-6 (1985)).

Gene name GenBankID Organism aspA P0AC38.1 Escherichia coli K12 ansBP26899.1 Bacillus subtilus aspA P07346.1 Pseudomonas fluorescens aspAP33109.1 Serratia marcescens

This example describes an MAA biosynthetic pathway from succinyl-CoA.

Example IV Preparation of an MAA Producing Microbial Organism Having aPathway for Converting Succinyl-CoA to MAA via3-Amino-2-methylpropanoate

This example describes the generation of a microbial organism capable ofproducing MAA from succinyl-CoA via 3-amino-2-methylpropanoate.

Escherichia coli is used as a target organism to engineer the MAApathway shown in FIG. 4. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing MAA. E. coliis amenable to genetic manipulation and is known to be capable ofproducing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions.

To generate an E. coli strain engineered to produce MAA, nucleic acidsencoding the enzymes utilized in the pathway are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the scpA(NP_(—)417392.1), argK (AAC75955.1), and AF454511 (AAL57846.1) genesencoding the methylmalonyl-CoA mutase, its stabilizer protein, andmethylmalonyl-CoA epimerase activities, respectively, are cloned intothe pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. In addition, the bphG (BAA03892.1), gabT (P22256.1), and aspA(POAC38.1) genes encoding methylmalonyl-CoA reductase,3-amino-2-methylpropionate transaminase, and 3-amino-2-methylpropionateammonia lyase activities, respectively, are cloned into the pZA33 vector(Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The twosets of plasmids are transformed into E. coli strain MG1655 to expressthe proteins and enzymes required for MAA synthesis via the succinyl-CoAto 3-amino-2-methylpropanoate pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the MAA synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce MAAis confirmed using HPLC, gas chromatography-mass spectrometry (GCMS)and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MAA synthesis pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of MAA. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of MAA. Adaptiveevolution also can be used to generate better producers of, for example,the succinyl-CoA intermediate of the MAA product. Adaptive evolution isperformed to improve both growth and production characteristics (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science314:1565-1568 (2006)). Based on the results, subsequent rounds ofmodeling, genetic engineering and adaptive evolution can be applied tothe MAA producer to further increase production.

For large-scale production of MAA, the above organism is cultured in afermenter using a medium known in the art to support growth of theorganism under anaerobic conditions. Fermentations are performed ineither a batch, fed-batch or continuous manner Anaerobic conditions aremaintained by first sparging the medium with nitrogen and then sealingthe culture vessel, for example, flasks can be sealed with a septum andcrimp-cap. Microaerobic conditions also can be utilized by providing asmall hole in the septum for limited aeration. The pH of the medium ismaintained at a pH of around 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce MAA from succinyl-CoA via 3-amino-2-methylpropanoate.

Example V Pathway for Conversion of 4-Hydroxybutyryl-CoA to3-Hydroxyisobutyric Acid or MAA

This example describes an exemplary 3-hydroxyisobutyric acid or MAAsynthesis pathway from 4-hydroxybutyryl-CoA.

An additional exemplary pathway entails the conversion of 4HB-CoA to MAA(see FIG. 6). In the first step, 4HB-CoA is converted to3-hydroxyisobutyryl-CoA (3-Hib-CoA) by a methylmutase. 3-Hib-CoA canthen be converted to 3-hydroxyisobutyrate by a CoA hydrolase, synthaseor transferase. 3-hydroxyisobutyrate can be secreted and recovered as aproduct or as a final step in the production of methacrylic acid.3-Hydroxybutyrate can be dehydrated to form methacrylic acid.Alternatively, 3-Hib-CoA can be dehydrated to methacrylyl-CoA which isthen converted to MAA by a hydrolase, synthase, or transferase. Theenzymes required for converting the tricarboxylic acid cycleintermediates, alpha-ketoglutarate, succinate, or succinyl-CoA, into4HB-CoA, are well-documented (Burk et al., U.S. application Ser. No.12/049,256, filed Mar. 14, 2008; Lutke-Eversloh and Steinbuchel. FEMSMicrobiol. Lett. 181:63-71 (1999); Sohling and Gottschalk, Eur. J.Biochem. 212:121-127 (1993); Sohling and Gottschalk, J. Bacteriol.178:871-880 (1996); Valentin et al., Eur. J. Biochem. 227:43-60 (1995);Wolff and Kenealy, Protein Expr. Purif. 6:206-212. (1995)).

Under anaerobic conditions, the maximum theoretical product yield is1.33 moles MAA per mol glucose if a CoA-transferase or synthetase isemployed to convert 3-hydroxyisobutyryl-CoA to 3-hydroxybutyrate in step2 of the pathway (Table 1). If a hydrolase is employed, the maximumtheoretical yield drops to 1.13 mol/mol unless PEP carboxykinase isassumed to reversibly operate in the ATP-generating direction towardsoxaloacetate. Likewise, the energetic yields are dependent on the typeof enzyme utilized in step 2 of the pathway. The highest ATP yields areobtained when a CoA-synthetase is utilized in step 2 and PEPcarboxykinase is assumed to be reversible. The product and energeticyields under aerobic conditions are also dependent on the type of enzymeutilized in the conversion of 3-hydroxyisobutyryl-CoA to3-hydroxybutyrate. It is understood that the maximum molar yields of ATPand product will be unchanged regardless of whether methacrylate or3-hydroxyisobutyrate is produced. Additionally, it is understood thatthe maximum molar yields of ATP and MAA will be unchanged if the pathwayproceeds through methacryl-CoA as depicted in FIG. 6.

TABLE 1 The maximum theoretical yield of MAA using the biosyntheticpathway through 4-hydroxybutyryl-CoA (4HB-CoA). All yields are expressedas mole/mole glucose. MAA biosynthetic pathway via 4HB-CoA AnaerobicAerobic MAA Yield (hydrolase for step 2 and/or 5) 1.13 1.28 MAA Yield(PEPCK reversible) 1.33 1.33 Max ATP yield @ max MAA yield 0.39 0.43(PEPCK reversible, hydrolase for step 2 and/or 5) Max ATP yield @ maxMAA yield 1.39 1.43 (PEPCK reversible, transferase for step 2 and/or 5)Max ATP yield @ max MAA yield 1.72 1.76 (PEPCK reversible, synthetasefor step 2 and/or 5)

Referring to FIG. 6, step 1 involves 4-hydroxybutyryl-CoA mutase (EC5.4.99.-). The conversion of 4HB-CoA to 3-hydroxyisobutyryl-CoA has yetto be demonstrated experimentally. However, two methylmutases, that is,isobutyryl-CoA mutase (ICM) and methylmalonyl-CoA mutase (MCM), whichcatalyze similar reactions, are good candidates given the structuralsimilarity of their corresponding substrates (FIG. 7). Methylmalonyl-CoAmutase is a cobalamin-dependent enzyme that converts succinyl-CoA tomethylmalonyl-CoA (FIG. 7A). This enzyme and suitable gene candidateswere discussed in the succinyl-CoA to MAA pathway (see Example I).

Alternatively, ICM could catalyze the proposed transformation. ICM is acobalamin-dependent methylmutase in the MCM family that reversiblyrearranges the carbon backbone of butyryl-CoA into isobutyryl-CoA (FIG.7B) (Ratnatilleke et al., J. Biol. Chem. 274:31679-31685 (1999)). Arecent study of a novel ICM in Methylibium petroleiphilum, along withprevious work, provides evidence that changing a single amino acid nearthe active site alters the substrate specificity of the enzyme(Ratnatilleke et al., J. Biol. Chem. 274:31679-31685 (1999); Rohwerderet al., Appl. Environ. Microbiol. 72:4128-4135. (2006)). This impliesthat if a native enzyme is unable to catalyze the conversion of 4HB-CoAto 3HIB-CoA, the enzyme could undergo rational engineering. ExemplaryICM genes encoding homodimeric enzymes include icmA in Streptomycescoelicolor A3 (Alhapel et al., Proc. Natl. Acad. Sci. USA103:12341-12346 (2006)) and Mpe_B0541 in Methylibium petroleiphilum PM1(Ratnatilleke et al., J. Biol. Chem. 274:31679-31685 (1999); Rohwerderet al., Appl. Environ. Microbiol. 72:4128-4135 (2006)). Genes encodingheterodimeric enzymes include icm and icmB in Streptomyces cinnamonensis(Ratnatilleke et al., J. Biol. Chem. 274:31679-31685 (1999); Vrijbloedet al., J. Bacteriol. 181:5600-5605. (1999); Zerbe-Burkhardt et al., J.Biol. Chem. 273:6508-6517 (1998)). Genes icmA and icmB in Streptomycesavermitilis MA-4680 show high sequence similarity to known ICMs.

Gene name GenBankID Organism icmA CAB40912.1 Streptomyces coelicolorA3(2) Mpe_B0541 YP_001023546.1 Methylibium petroleiphilum PM1 icmAAC08713.1 Streptomyces cinnamonensis icmB CAB59633.1 Streptomycescinnamonensis icmA NP_824008.1 Streptomyces avermitilis MA-4680 icmBNP_824637.1 Streptomyces avermitilis MA-4680

Referring to FIG. 6, step 2 involves 3-hydroxyisobutyryl-CoA hydrolase(EC 3.1.2.4), synthetase (EC 6.2.1.-) or 3-hydroxyisobutyryl-CoAtransferase (EC 2.8.3.-). Step 5 involves methacrylyl-CoA hydrolase,synthetase, or transferase. These transformations can be performed bydifferent classes of enzymes including CoA hydrolases (EC 3.1.2.-), CoAtransferases (EC 2.8.3.-), and CoA synthetases (EC 6.1.2.-). Asdiscussed earlier, pathway energetics are most favorable if a CoAtransferase or a CoA synthetase is employed to accomplish thistransformation (Table 1).

In the CoA-transferase family, E. coli enzyme acyl-CoA:acetate-CoAtransferase, also known as acetate-CoA transferase (EC 2.8.3.8), hasbeen shown to transfer the CoA moity to acetate from a variety ofbranched and linear acyl-CoA substrates, including isobutyrate (Matthiesand Schink, Appl. Environ. Microbiol. 58:1435-1439 (1992)), valerate(Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968))and butanoate (Vanderwinkel et al. supra, 1968). This enzyme is encodedby atoA (alpha subunit) and atoD (beta subunit) in E. coli sp. K12(Korolev et al., Acta Crystallogr. D Biol. Crystallogr. 58:2116-2121(2002); Vanderwinkel et al., supra, 1968) and actA and cg0592 inCorynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ.Microbiol. 68:5186-5190 (2002)) and represents an ideal candidate tocatalyze the desired 3-hydroxyisobutyryl-CoA transferase ormethacrylyl-CoA transferase biotransformations shown in FIG. 6, steps 2and 5. Candidate genes by sequence homology include atoD and atoA inEscherichia coli UT189. Similar enzymes also exist in Clostridiumacetobutylicum and Clostridium saccharoperbutylacetonicum.

Gene name GenBankID Organism atoA P76459.1 Escherichia coli K12 atoDP76458.1 Escherichia coli K12 actA YP_226809.1 Corynebacteriumglutamicum ATCC 13032 cg0592 YP_224801.1 Corynebacterium glutamicum ATCC13032 atoA ABE07971.1 Escherichia coli UT189 atoD ABE07970.1 Escherichiacoli UT189 ctfA NP_149326.1 Clostridium acetobutylicum ctfB NP_149327.1Clostridium acetobutylicum ctfA AAP42564.1 Clostridiumsaccharoperbutylacetonicum ctfB AAP42565.1 Clostridiumsaccharoperbutylacetonicum

Additional exemplary transferase transformations are catalyzed by thegene products of cat1, cat2, and cat3 of Clostridium kluyveri which havebeen shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, andbutyryl-CoA acetyltransferase activity, respectively (Sohling andGottschalk, J. Bacteriol. 178 (3): 871-880 (1996); Seedorf et al., Proc.Natl. Acad. Sci. USA, 105 (6):2128-2133 (2008)).

Gene name GenBankID Organism cat1 P38946.1 Clostridium kluyveri cat2P38942.2 Clostridium kluyveri cat3 EDK35586.1 Clostridium kluyveri

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack and Buckel, FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51(1994)).

Gene name GenBankID Organism gctA CAA57199.1 Acidaminococcus fermentansgctB CAA57200.1 Acidaminococcus fermentans

Additional enzyme candidates include succinyl-CoA:3-ketoacid CoAtransferases which utilize succinate as the CoA acceptor. Exemplarysuccinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacterpylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997))and Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403(2007)).

Gene name GenBankID Organism HPAG1_0676 YP_627417 Helicobacter pyloriHPAG1_0677 YP_627418 Helicobacter pylori ScoA NP_391778 Bacillussubtilis ScoB NP_391777 Bacillus subtilis

A candidate ATP synthase is ADP-forming acetyl-CoA synthetase (ACD, EC6.2.1.13), an enzyme that couples the conversion of acyl-CoA esters totheir corresponding acids with the concurrent synthesis of ATP. Althoughthis enzyme has not been shown to react with 3-hydroxyisobutyryl-CoA ormethacrylyl-CoA as a substrate, several enzymes with broad substratespecificities have been described in the literature. ACD I fromArchaeoglobus fulgidus, encoded by AF1211, was shown to operate on avariety of linear and branched-chain substrates including isobutyrate,isopentanoate, and fumarate (Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotatedas a succinyl-CoA synthetase) accepts propionate, butyrate, andbranched-chain acids (isovalerate and isobutyrate) as substrates, andwas shown to operate in the forward and reverse directions (Brasen andSchonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded byPAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilumshowed the broadest substrate range of all characterized ACDs, reactingwith acetyl-CoA, isobutyryl-CoA (preferred substrate) andphenylacetyl-CoA (Brasen and Schonheit, supra, 2004). However, directedevolution or engineering can be used to modify this enzyme to operate atthe physiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned,functionally expressed, and characterized in E. coli (Brasen andSchonheit, supra, 2004; Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)).

Gene name GenBankID Organism AF1211 NP_070039.1 Archaeoglobus fulgidusDSM 4304 scs YP_135572.1 Haloarcula marismortui ATCC 43049 PAE3250NP_560604.1 Pyrobaculum aerophilum str. IM2

In the CoA hydrolase family, the enzyme 3-hydroxyisobutyryl-CoAhydrolase is specific for 3-HIBCoA and has been described to efficientlycatalyze the desired transformation during valine degradation (Shimomuraet al., J. Biol. Chem. 269:14248-14253 (1994)). Genes encoding thisenzyme include hibch of Rattus norvegicus (Shimomura et al., J. Biol.Chem. 269:14248-14253 (1994); Shimomura et al., Methods Enzymol.324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra, 2000).Candidate genes by sequence homology include hibch of Saccharomycescerevisiae and BC 2292 of Bacillus cereus.

Gene name GenBankID Organism hibch Q5XIE6.2 Rattus norvegicus hibchQ6NVY1.2 Homo sapiens hibch P28817.2 Saccharomyces cerevisiae BC_2292Q81DR3 Bacillus cereus

Referring to FIG. 6, step 3 involves 3-hydroxyisobutyrate dehydratase(EC 4.2.1.-). The entails dehydration of 3-hydroxyisobutyrate to MAA by3-hydroxyisobutyrate dehydratase. Gene candidates for this enzyme aredescribed in the succinyl-CoA to MAA pathway (see Example I). Alsoreferring to FIG. 6, step 4 involves 3-hydroxyisobutyryl-CoA dehydratase(EC 4.2.1.-). Dehydration of 3-hydroxyisobutyryl-CoA to methacrylyl-CoAcan be accomplished by a reversible 3-hydroxyacyl-CoA dehydratase suchas crotonase (also called 3-hydroxybutyryl-CoA dehydratase, EC 4.2.1.55)or enoyl-CoA hydratase (also called 3-hydroxyacyl-CoA dehydratase, EC4.2.1.17). These enzymes are generally reversible (Moskowitz andMerrick, Biochemistry 8:2748-2755 (1969); Dune et al., FEMS Microbiol.Rev. 17:251-262 (1995)). Exemplary genes encoding crotonase enzymes canbe found in C. acetobutylicum (Boynton, et al., J. Bacteriol. 178(11):3015-3024 (1996)), C. kluyveri (Hillmer and Gottschalk, FEBS Lett.21 (3):351-354 (1972)), and Metallosphaera sedula (Berg et al., Science318 (5857) 1782-1786 (2007)) though the sequence of the latter gene isnot known. Enoyl-CoA hydratases, which are involved in fatty acidbeta-oxidation and/or the metabolism of various amino acids, can alsocatalyze the hydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA(Agnihotri and Liu, Bioorg. Med. Chem. 11 (1):9-20 (2003); Roberts etal., Arch. Microbiol. 117 (1):99-108 (1978); Conrad et al., J.Bacteriol. 118 (1):103-111 (1974)). The enoyl-CoA hydratases, phaA andphaB, of P. putida are believed to carry out the hydroxylation of doublebonds during phenylacetate catabolism (Olivera et al., Proc. Natl. Acad.Sci. USA 95:6419-6424 (1998)). The paaA and paaB from P. fluorescenscatalyze analogous transformations (Olivera et al., supra, 1998).Lastly, a number of Escherichia coli genes have been shown todemonstrate enoyl-CoA hydratase functionality including maoC (Park andLee, J. Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., Eur. J.Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol.113-116:335-346 (2004); Park and Yup, Biotechnol. Bioeng. 86:681-686.(2004)), and paaG (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003);Park and Lee, Appl. Biochem. Biotechnol. 113-116:335-346 (2004); Parkand Yup, Biotechnol. Bioeng. 86:681-686 (2004)).

Gene name GenBankID Organism crt NP_349318.1 Clostridium acetobutylicumcrt1 YP_001393856 Clostridium kluyveri DSM 555 paaA NP_745427.1Pseudomonas fluorescens paaB NP_745426.1 Pseudomonas fluorescens phaAABF82233.1 Pseudomonas putida phaB ABF82234.1 Pseudomonas putida maoCNP_415905.1 Escherichia coli paaF NP_415911.1 Escherichia coli paaGNP_415912.1 Escherichia coli

This example describes a biosynthetic pathway for production of3-hydroxyisobutyric acid or methacrylic acid from 4-hydroxybutyryl-CoA.

Example VI Preparation of an MAA Producing Microbial Organism Having aPathway for Converting 4-hydroxybutyryl-CoA to MAA

This example describes the generation of a microbial organism capable ofproducing MAA from 4-hydroxybutyryl-CoA.

Escherichia coli is used as a target organism to engineer the MAApathway shown in FIG. 6. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing MAA. E. coliis amenable to genetic manipulation and is known to be capable ofproducing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions.

To generate an E. coli strain engineered to produce MAA, nucleic acidsencoding the enzymes utilized in the pathway are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the sucD(YP_(—)001396394), 4hbd (YP_(—)001396393), buk1 (Q45829), and ptb(NP_(—)349676) genes encoding succinic semialdehyde dehydrogenase(CoA-dependent), 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyratekinase, and phosphotransbutyrylase activities, respectively, are clonedinto the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. This construct allows the production of 4HB-CoA fromsuccinyl-CoA as described in Burk et al. (U.S. application Ser. No.12/049,256, filed Mar. 14, 2008). In addition, the icmA (CAB40912.1),hibch (Q5XIE6.2), and hmd (ABC88407.1) genes encoding4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA hydrolase, and3-hydroxyisobutyrate dehydratase activities, respectively, are clonedinto the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. The two sets of plasmids are transformed into E. coli strainMG1655 to express the proteins and enzymes required for MAA synthesisvia the 4-hydroxybutyryl-CoA pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the MAA synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce MAAis confirmed using HPLC, gas chromatography-mass spectrometry (GCMS)and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MAA synthesis pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of MAA. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of MAA. Adaptiveevolution also can be used to generate better producers of, for example,the succinyl-CoA or 4-hydroxybutyryl-CoA intermediates of the MAAproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the MAA producer to further increaseproduction.

For large-scale production of MAA, the above organism is cultured in afermenter using a medium known in the art to support growth of theorganism under anaerobic conditions. Fermentations are performed ineither a batch, fed-batch or continuous manner Anaerobic conditions aremaintained by first sparging the medium with nitrogen and then sealingthe culture vessel, for example, flasks can be sealed with a septum andcrimp-cap. Microaerobic conditions also can be utilized by providing asmall hole in the septum for limited aeration. The pH of the medium ismaintained at a pH of around 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce MAA from 4-hydroxybutyryl-CoA.

Example VII Pathway for Conversion of Alpha-ketoglutarate to MAA viaThreo-3-methylaspartate

This example describes an exemplary MAA synthesis pathway fromalpha-ketoglutarate to threo-3-methylaspartate.

Another exemplary pathway for MAA biosynthesis proceeds throughalpha-ketoglutarate, a metabolite in E. coli produced in the TCA cycle(see FIG. 8). This pathway is high-yielding under aerobic conditionswith a maximum theoretical yield of 1.2 mol MAA/mol glucose (Table 2).The yields under anaerobic conditions are lower, as the pathway is redoximbalanced and MAA synthesis requires the formation of fermentationbyproducts such as formate and ethanol.

TABLE 2 The maximum theoretical yield of MAA using thealpha-ketoglutarate biosynthetic pathway. All yields are expressed asmole/mole glucose. MAA biosynthetic pathway via alpha- ketoglutarateAnaerobic Aerobic MAA Yield 0.69 1.2 MAA Yield (PEPCK reversible) 0.821.2 ATP Yield @ max MAA yield (PEPCK 0 0.95 reversible)

The first step of the pathway, catalyzed by the enzyme aspartateaminotransferase, transfers an amino group from aspartate toalpha-ketoglutarate, forming glutamate and oxaloacetate. The subsequenttwo steps include rearrangement of the carbon backbone and subsequentdeamination to form mesaconate. Enzymes catalyzing these conversions arefound in the energy-yielding fermentation of glutamate in soilClostridia and other organisms capable of fermenting amino acids (Buckeland Barker, J. Bacteriol. 117:1248-1260 (1974)). The directionality ofthe pathway in these organisms is in agreement with the directionrequired for MAA synthesis in the biopathway. The final pathway stepentails decarboxylation of mesaconate to yield methacrylic acid.

Referring to FIG. 8, step 1 involves aspartate aminotransferase (EC2.6.1.1). The first step of the pathway transfers an amino group fromaspartate to alpha-ketoglutarate, forming glutamate and oxaloacetate.The genes aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84(1979); Yagi et al., Methods Enzymol. 113:83-89 (1985)), AAT2 fromSaccharomyces cerevisiae (Yagi et al., J. Biochem. 92:35-43 (1982)) andASPS from Arabidopsis thaliana (de la Torre et al., Plant J. 46:414-425(2006); Kwok and Hanson, J. Exp. Bot. 55:595-604 (2004); Wilkie andWarren, Protein Expr. Purif. 12:381-389 (1998)), encode the enzyme thatcatalyzes this conversion, aspartate aminotransferase.

Gene name GenBank Accession # Organism aspC NP_415448.1 Escherichia coliAAT2 P23542.3 Saccharomyces cerevisiae ASP5 P46248.2 Arabidopsisthaliana

Referring to FIG. 8, step 2 involves glutamate mutase (EC 5.4.99.1). Instep 2, the linear carbon chain of glutamate is rearranged to thebranched structure of threo-3-methylaspartate. This transformation iscatalyzed by glutamate mutase, a cobalamin-dependent enzyme composed oftwo subunits. Two glutamate mutases, from Clostridium cochlearium andClostridium tetanomorphum, have been cloned and functionally expressedin E. coli (Holloway and Marsh, J. Biol. Chem. 269:20425-20430 (1994);Reitzer et al., Acta Crystallogr. D Biol. Crystallogr. 54:1039-1042(1998)). The genes encoding this two-subunit protein are glmE and glmSfrom Clostridium cochlearium, mamA and glmE from Clostridiumtetanomorphum, and mutE and mutS from Clostridium tetani (Switzer,Glutamate mutase, pp. 289-305 Wiley, New York (1982)).

Gene name GenBankID Organism glmE P80077.2 Clostridium cochlearium glmSP80078.2 Clostridium cochlearium mamA Q05488.1 Clostridium tetanomorphumglmE Q05509.1 Clostridium tetanomorphum mutE NP_783086.1 Clostridiumtetani E88 mutS NP_783088.1 Clostridium tetani E88

Referring to FIG. 8, step 3 involves 3-methylaspartase (EC 4.3.1.2).3-methylaspartase, also referred to as beta-methylaspartase or3-methylaspartate ammonia-lyase, catalyzes the deamination ofthreo-3-methylasparatate to mesaconate. The 3-methylaspartase fromClostridium tetanomorphum has been cloned, functionally expressed in E.coli, and crystallized (Asuncion et al., Acta Crystallogr. D Biol.Crystallogr. 57:731-733 (2001); Asuncion et al., J. Biol. Chem.277:8306-8311 (2002); Botting et al., Biochemistry 27:2953-2955 (1988);Goda et al., Biochemistry 31:10747-10756 (1992)). In Citrobacteramalonaticus, this enzyme is encoded by BAA28709 (Kato and Asano, Arch.Microbiol. 168:457-463 (1997)). 3-methylaspartase has also beencrystallized from E. coli YG1002 (Asano and Kato, FEMS Microbiol. Lett.118:255-258 (1994)), although the protein sequence is not listed inpublic databases such as GenBank. Sequence homology can be used toidentify additional candidate genes, including CTC 02563 in C. tetaniand ECs0761 in Escherichia coli O157:H7.

Gene name GenBankID Organism MAL AAB24070.1 Clostridium tetanomorphumBAA28709 BAA28709.1 Citrobacter amalonaticus CTC_02563 NP_783085.1Clostridium tetani ECs0761 BAB34184.1 Escherichia coli O157:H7 str.Sakai

Referring to FIG. 8, step 4 involves mesaconate decarboxylase (EC4.1.1.-). The final step of the pathway entails the decarboxylation ofmesaconate to methacrylic acid. An enzyme catalyzing this exact reactionhas not been demonstrated experimentally. However, several enzymescatalyzing highly similar reactions exist (FIG. 9). One enzyme withclosely related function is aconitate decarboxylase (FIG. 9A). Thisenzyme catalyzes the final step in itaconate biosynthesis in a strain ofCandida and the filamentous fungi Aspergillus terreus (Bonnarme et al.,J. Bacteriol. 177:3573-3578 (1995); Willke and Vorlop, Appl. Microbiol.Biotechnol. 56:289-295 (2001)). Although itaconate is a compound ofbiotechnological interest, no efforts have been made thus far toidentify or clone the aconitate decarboxylase gene.

A second enzyme with similar function is 4-oxalocronate decarboxylase(FIG. 9B). This enzyme is common in a variety of organisms and the genesencoding the enzyme from Pseudomonas sp. (strain 600) have been clonedand expressed in E. coli (Shingler et al., J. Bacteriol. 174:711-724(1992)). The methyl group in mesaconate may cause steric hindrance, butthis problem could likely be overcome with directed evolution or proteinengineering. 4-oxalocronate decarboxylase is composed of two subunits.Genes encoding this enzyme include dmpH and dmpE in Pseudomonas sp.(strain 600) (Shingler et al., J. Bacteriol. 174:711-724 (1992)), xylIIand xylIII from Pseudomonas putida (Kato and Asano, Arch. Microbiol.168:457-463 (1997); Stanley et al., Biochemistry 39:718-726 (2000)), andReut_B5691 and Reut_B5692 from Ralstonia eutropha JMP134 (Hughes et al.,J. Bacteriol. 158:79-83 (1984)).

Gene name GenBankID Organism dmpH CAA43228.1 Pseudomonas sp. CF600 dmpECAA43225.1 Pseudomonas sp. CF600 xylII YP_709328.1 Pseudomonas putidaxylIII YP_709353.1 Pseudomonas putida Reut_B5691 YP_299880.1 Ralstoniaeutropha JMP134 Reut_B5692 YP_299881.1 Ralstonia eutropha JMP134

This example describes a biosynthetic pathway for production of MMA fromalpha-ketoglutarate.

Example VIII Preparation of an MAA Producing Microbial Organism Having aPathway for Converting Alpha-ketoglutarate to MAA viaThreo-3-Methylaspartate

This example describes the generation of a microbial organism capable ofproducing MAA from alpha-ketoglutarate via threo-3-methylaspartate.

Escherichia coli is used as a target organism to engineer the MAApathway shown in FIG. 8. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing MAA. E. coliis amenable to genetic manipulation and is known to be capable ofproducing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions.

To generate an E. coli strain engineered to produce MAA, nucleic acidsencoding the enzymes utilized in the pathway are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the aspC(NP_(—)415448.1), glmE (P80077.2), and glmS (P80078.2) genes encodingthe aspartate aminotransferase and glutamate mutase activities arecloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under thePA1/lacO promoter. In addition, the MAL (AAB24070.1), dmpH (CAA43228.1),and dmpE (CAA43225.1) genes encoding 3-methylaspartase and mesaconatedecarboxylase activities are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. The two sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for MAA synthesis via thealpha-ketoglutarate to threo-3-methylaspartate pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the MAA synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce MAAis confirmed using HPLC, gas chromatography-mass spectrometry (GCMS)and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MAA synthesis pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of MAA. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of MAA. Adaptiveevolution also can be used to generate better producers of, for example,the alpha-ketoglutarate intermediate of the MAA product. Adaptiveevolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the MAA producer to further increaseproduction.

For large-scale production of MAA, the above organism is cultured in afermenter using a medium known in the art to support growth of theorganism under anaerobic conditions. Fermentations are performed ineither a batch, fed-batch or continuous manner Anaerobic conditions aremaintained by first sparging the medium with nitrogen and then sealingthe culture vessel, for example, flasks can be sealed with a septum andcrimp-cap. Microaerobic conditions also can be utilized by providing asmall hole in the septum for limited aeration. The pH of the medium ismaintained at a pH of around 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce MAA from alpha-ketoglutarate via threo-3-methylaspartate.

Example IX Pathway for Conversion of Alpha-ketoglutarate to MAA via2-Hydroxyglutarate

This example describes an exemplary MAA synthesis pathway fromalph-ketoglutarate to MAA via 2-hydroxyglutarate.

Another exemplary pathway for MAA biosynthesis has a scheme similar tothe pathway described in Example VII, but it passes through thehydroxylated intermediates 2-hydroxyglutarate and 3-methylmalate (seeFIG. 10), rather than amine-substituted intermediates (see FIG. 8). Likethe pathway described in Example VII, this pathway is high-yieldingunder aerobic conditions with a maximum theoretical yield of 1.2 molMAA/mol glucose (Table 3). Under anaerobic conditions, the pathway isnot redox-balanced and MAA synthesis requires formation of fermentationbyproducts such as ethanol, formate and succinate.

TABLE 3 The maximum theoretical yield of MAA using thealpha-ketoglutarate biosynthetic pathway via 2-hydroxyglutarate. Allyields are expressed as mole/mole glucose. MAA biosynthetic pathway viaalpha- ketoglutarate (alt) Anaerobic Aerobic MAA Yield 0.74 1.20 MAAYield (PEPCK reversible) 0.87 1.20 ATP Yield @ max MAA yield (PEPCK 01.55 reversible)

Referring to FIG. 10, step 1 involves alpha-ketoglutarate reductase (EC1.1.99.2). The first step of this pathway entails the reduction ofalpha-ketoglutarate to 2-hydroxyglutarate by native enzymealpha-ketoglutarate reductase. This enzyme is encoded by serA, amultifunctional enzyme which also catalyzes the reduction of3-phosphoglycerate in central metabolism (Zhao and Winkler, J.Bacteriol. 178:232-239 (1996)). Genes L2HGDH in Homo sapiens (Jansen andWanders, Biochim. Biophys. Acta 1225:53-56 (1993)), FN0487 in L2hgdh inFusobacterium nucleatum (Hayashi et al., J. Nihon Univ. Sch. Dent.28:12-21 (1986)), and L2hgdh_predicted in Rattus norvegicus (Jansen andWanders, Biochim. Biophys. Acta 1225:53-56 (1993)) encode this enzyme.Gene candidates with high sequence homology include L2hgdh in Musmusculus and L2HGDH in Bos taurus. At high concentrations,2-hydroxyglutarate has been shown to feed back on alpha-ketoglutaratereductase activity by competitive inhibition (Zhao and Winkler, J.Bacteriol. 178:232-239. (1996)).

Gene name GenBankID Organism serA CAA01762.1 Escherichia coli L2HGDHQ9H9P8.2 Homo sapiens L2hgdh NP_663418.1 Mus musculus L2hgdh_predictedNP_001101498.1 Rattus norvegicus L2HGDH NP_001094560.1 Bos taurus FN0487Q8RG31 Fusobacterium nucleatum subsp. Nucleatum

Referring to FIG. 10, step 2 involves 2-hydroxyglutamate mutase (EC5.4.99.-). In the second step of the pathway, the carbon backboneundergoes rearrangement by a glutamate mutase enzyme. The most commonreaction catalyzed by such an enzyme is the conversion of glutamate tothreo-3-methylasparate, shown in step 2 of FIG. 8. Theadenosylcobalamin-dependent glutamate mutase from Clostridiumcochlearium has also been shown to react with 2-hydroxyglutarate as analternate substrate (Roymoulik et al., Biochemistry 39:10340-10346(2000)), although the rate of this reaction is two orders of magnitudelower with 2-hydroxyglutarate compared to the rate with native substrateglutamate. Directed evolution of the enzyme can be used to increaseglutamate mutase affinity for 2-hydroxyglutarate. GenBank accessionnumbers of protein sequences encoding glutamate mutases are found inExample VII, step 2 of the pathway.

Referring to FIG. 10, step 3 involves 3-methylmalate dehydratase (EC4.2.1.-). In the third step, 3-methylmalate is dehydrated to formmesaconate. Although an enzyme catalyzing this exact transformation hasnot been described in the literature, several enzymes are able tocatalyze a similar reaction (FIG. 11). One such enzyme is 2-methylmalatedehydratase, also called citramalate hydrolyase, which converts2-methylmalate to mesaconate (FIG. 11A). 2-Methylmalate and3-methylmalate are closely related, with the only difference instructure being the location of the hydroxyl group. 2-Methylmalatedehydratase activity was detected in Clostridium tetanomorphum,Morganella morganii, Citrobacter amalonaticus in the context of theglutamate degradation VI pathway (Kato and Asano, Arch. Microbiol.168:457-463 (1997)); however the genes encoding this enzyme have notbeen sequenced to date.

A second candidate enzyme is fumarate hydratase, which catalyzes thedehydration of malate to fumarate (FIG. 11B). As described in Example I(step 5), a wealth of structural information is available for thisenzyme and other studies have successfully engineered the enzyme toalter activity, inhibition and localization (Weaver, Acta Crystallogr. DBiol. Crystallogr. 61:1395-1401 (2005)). Gene candidates are discussedin Example I, step 5 of the pathway.

Referring to FIG. 10, step 4 involves mesaconate decarboxylase (EC4.1.1.-). The final pathway step involves the decarboxylation ofmesaconate to methacrylic acid. This reaction is identical to the finalstep of the pathway described in Example VII.

This example describes a biosynthetic pathway for production of MMA fromalpha-ketoglutarate.

Example X Preparation of an MAA Producing Microbial Organism Having aPathway for Converting Alpha-ketoglutarate to MAA via 2-Hydroxyglutarate

This example describes the generation of a microbial organism capable ofproducing MAA from alpha-ketoglutarate via 2-hydroxyglutarate.

Escherichia coli is used as a target organism to engineer the MAApathway shown in FIG. 10. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing MAA. E. coliis amenable to genetic manipulation and is known to be capable ofproducing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions.

To generate an E. coli strain engineered to produce MAA, nucleic acidsencoding the enzymes utilized in the pathway are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the serA(CAA01762.1), glmE (P80077.2), and glmS (P80078.2) genes encoding thealpha-ketoglutarate reductase and 2-hydroxyglutamate mutase activitiesare cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) underthe PA1/lacO promoter. In addition, the fumC (P05042.1), dmpH(CAA43228.1), and dmpE (CAA43225.1) genes encoding 3-methylmalatedehydratase and mesaconate decarboxylase activities are cloned into thepZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. The two sets of plasmids are transformed into E. coli strainMG1655 to express the proteins and enzymes required for MAA synthesisvia the alpha-ketoglutarate to 2-hydroxyglutarate pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the MAA synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce MAAis confirmed using HPLC, gas chromatography-mass spectrometry (GCMS)and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MAA synthesis pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of MAA. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of MAA. Adaptiveevolution also can be used to generate better producers of, for example,the alpha-ketoglutarate intermediate of the MAA product. Adaptiveevolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the MAA producer to further increaseproduction.

For large-scale production of MAA, the above organism is cultured in afermenter using a medium known in the art to support growth of theorganism under anaerobic conditions. Fermentations are performed ineither a batch, fed-batch or continuous manner Anaerobic conditions aremaintained by first sparging the medium with nitrogen and then sealingthe culture vessel, for example, flasks can be sealed with a septum andcrimp-cap. Microaerobic conditions also can be utilized by providing asmall hole in the septum for limited aeration. The pH of the medium ismaintained at a pH of around 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce MAA from alpha-ketoglutarate via 2-hydroxyglutarate.

Example XI Pathway for Conversion of Acetyl-CoA to 2-HydroxyisobutyricAcid or MAA

This example describes an exemplary 2-hydroxyisobutyric acid or MAAsynthesis pathway from acetyl-CoA.

MAA biosynthesis can proceed from acetyl-CoA in a minimum of fiveenzymatic steps (see FIG. 12). In this pathway, two molecules ofacetyl-CoA are combined to form acetoacetyl-CoA, which is then reducedto 3-hydroxybutyryl-CoA. Alternatively, 4-hydroxybutyryl-CoA can beconverted to 3-hydroxybutyryl-CoA by way of 4-hydroxybutyryl-CoAdehydratase and crotonase (Martins et al., Proc. Nat. Acad. Sci. USA 101(44) 15645-15649 (2004); Jones and Woods, Microbiol. Rev. 50:484-524(1986); Berg et al., Science 318 (5857) 1782-1786 (2007)). Amethylmutase then rearranges the carbon backbone of 3-hydroxybutyryl-CoAto 2-hydroxyisobutyryl-CoA, which is then dehydrated to formmethacrylyl-CoA. Alternatively, 2-hydroxyisobutyryl-CoA can be convertedto 2-hydroxyisobutyrate, secreted, and recovered as product. The finalstep converting methacrylyl-CoA to MAA can be performed by a singleenzyme (shown in FIG. 12) or a series of enzymes.

The pathway shown in FIG. 12 has a maximum theoretical product yield of1.25 mol/mol glucose under aerobic conditions which also requires theutilization of 0.4 moles of oxygen (Table 4). In the absence of oxygenuptake, the maximum theoretical yield drops to 1.01 mol/mol glucose andthe fermentation byproducts such as ethanol and formate must be formedto maintain redox balance. The assumption that PEP carboxykinase (PEPCK)can operate in the ATP generating direction increases the MAA yieldunder anaerobic conditions to 1.09 mol/mol, but does not prevent theformation of byproducts. The energetics of MAA formation are favorableif a CoA transferase or synthetase is utilized in step 5 of the pathway.Equivalent maximum yields of product and ATP are obtain if2-hydroxyisobutyric acid is produced as opposed to methacrylic acid viathe pathways described herein.

TABLE 4 MAA and ATP yields for Acetyl-CoA pathway. MAA biosyntheticpathway via Acetyl-CoA Anaerobic Aerobic MAA Yield 1.01 1.25 MAA Yield(PEPCK reversible) 1.09 1.25 Max ATP yield @ max MAA yield 0 0.03 (PEPCKreversible, hydrolase for step 5) Max ATP yield @ max MAA yield 1.091.28 (PEPCK reversible, transferase or synthetase for step 5)

Referring to FIG. 12, step 1 involves acetoacetyl-CoA thiolase (EC2.3.1.9). The formation of acetoacetyl-CoA from two acetyl-CoA units iscatalyzed by acetyl-CoA thiolase. This enzyme is native to E. coli,encoded by gene atoB, and typically operates in theacetoacetate-degrading direction during fatty acid oxidation (Duncombeand Frerman, Arch. Biochem. Biophys. 176:159-170 (1976); Frerman andDuncombe, Biochim. Biophys. Acta 580:289-297 (1979)). The gene thlA fromClostridium acetobutylicum was engineered into an isopropanol-producingstrain of E. coli and was shown to function in the direction ofacetoacetate synthesis (Hanai et al., Appl. Environ. Microbiol.73:7814-7818 (2007); Stim-Herndon et al., Gene 154:81-85 (1995)). Anadditional gene candidate is thl from Clostridium pasteurianum (Meng andLi. Cloning, Biotechnol. Lett. 28:1227-1232 (2006)).

Gene name GenBankID Organism atoB P76461.1 Escherichia coli thlAP45359.1 Clostridium acetobutylicum thl ABA18857.1 Clostridiumpasteurianum

Referring to FIG. 12, step 2 involves acetoacetyl-CoA reductase (EC#:1.1.1.35). The second step entails the reduction of acetoacetyl-CoA to3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase. This enzymeparticipates in the acetyl-CoA fermentation pathway to butyrate inseveral species of Clostridia and has been studied in detail (Jones andWoods, Microbiol. Rev. 50:484-524 (1986)). The enzyme from Clostridiumacetobutylicum, encoded by hbd, has been cloned and functionallyexpressed in E. coli (Youngleson et al., J. Bacteriol. 171:6800-6807(1989)). Additionally, subunits of two fatty acid oxidation complexes inE. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoAdehydrogenases (Binstock and Schulz, Methods Enzymol. 71 Pt C:403-411(1981)). Additional gene candidates include Hbd1 (C-terminal domain) andHbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer andGottschalk, Biochim. Biophys. Acta 3334:12-23 (1974)) and HSD17B10 inBos taurus (Wakil et al., J. Biol. Chem. 207:631-638 (1954)).

Gene name GenBankID Organism fadB P21177.2 Escherichia coli fadJP77399.1 Escherichia coli Hbd2 EDK34807.1 Clostridium kluyveri Hbd1EDK32512.1 Clostridium kluyveri hbd P52041.2 Clostridium acetobutylicumHSD17B10 O02691.3 Bos taurus

Referring to FIG. 12, step 3 involves 3-hydroxybutyryl-CoA mutase (EC5.4.99.-). In the next step, 3-hydroxybutyryl-CoA, is rearranged to form2-HIBCoA by 3-hydroxybutyryl-CoA mutase. This enzyme is a novel ICM-likemethylmutase recently discovered and characterized in Methylibiumpetroleiphilum (Ratnatilleke et al., J. Biol. Chem. 274:31679-31685(1999); Rohwerder et al., Appl. Environ. Microbiol. 72:4128-4135(2006)). This enzyme, encoded by Mpe_B0541 in Methylibium petroleiphilumPM1, has high sequence homology to the large subunit ofmethylmalonyl-CoA mutase in other organisms including Rsph17029_(—)3657in Rhodobacter sphaeroides and Xaut_(—)5021 in Xanthobacterautotrophicus. As discussed in Example V (step 1), changes to a singleamino acid near the active site alters the substrate specificity of theenzyme (Ratnatilleke et al., supra, 1999; Rohwerder et al., supra,2006), so alternate gene candidates for this enzyme can be engineered atthis site to achieve the appropriate reactivity.

Gene name GenBankID Organism Mpe_B0541 YP_001023546.1 Methylibiumpetroleiphilum PM1 Rsph17029_3657 YP_001045519.1 Rhodobacter sphaeroidesATCC 17029 Xaut_5021 YP_001409455.1 Xanthobacter autotrophicus Py2

Referring to FIG. 12, step 4 involves 2-hydroxyisobutyryl-CoAdehydratase. The dehydration of 2-hydroxyacyl-CoA can be catalyzed by aspecial class of oxygen-sensitive enzymes that operate via aradical-mechanism (Buckel and Golding, Annu. Rev. Microbiol. 60:27-49(2006); Buckel et al., Curr. Opin. Chem. Biol. 8:462-467 (2004); Buckelet al., Biol. Chem. 386:951-959 (2005); Kim et al., FEBS J. 272:550-561(2005); Kim et al., FEMS Microbiol. Rev. 28:455-468 (2004); Zhang etal., Microbiology 145 (Pt 9):2323-2334 (1999)). One example of such anenzyme is the lactyl-CoA dehydratase from Clostridium propionicum, whichcatalyzes the dehydration of lactoyl-CoA to form acryl-CoA (Kuchta andAbeles, J. Biol. Chem. 260:13181-13189 (1985); Hofineister and Buckel,Eur. J. Biochem. 206:547-552 (1992)). An additional example is2-hydroxyglutaryl-CoA dehydratase encoded by hgdABC from Acidaminococcusfermentans (Muëller and Buckel, Eur. J. Biochem. 230:698-704 (1995);Schweiger et al., Eur. J. Biochem. 169:441-448 (1987)). Yet anotherexample is the 2-hydroxyisocaproyl-CoA dehydratase from Clostridiumdifficile catalyzed by hadBC and activated by hadI (Darley et al., FEBSJ. 272:550-61 (2005)). The corresponding sequences for A. fermentans andC. difficile can be found using the following GenBankIDs, while thesequences for C. propionicium are not yet listed in publicly availabledatabases.

Gene name GenBankID Organism hgdA P11569 Acidaminococcus fermentans hgdBP11570 Acidaminococcus fermentans hgdC P11568 Acidaminococcus fermentanshadB YP_001086863 Clostridium difficile hadC YP_001086864 Clostridiumdifficile hadI YP_001086862 Clostridium difficile

Referring to FIG. 12, steps 5 or 6 involve a transferase (EC 2.8.3.-),hydrolase (EC 3.1.2.-), or synthetase (EC 6.2.1.-) with activity on amethacrylic acid or 2-hydroxyisobutyric acid, respectively. Directconversion of methacrylyl-CoA to MAA or 2-hydroxyisobutyryl-CoA to2-hydrioxyisobutyrate can be accomplished by a CoA transferase,synthetase or hydrolase. As discussed in Example V, pathway energeticsare most favorable if a CoA transferase or a CoA synthetase is employedto accomplish this transformation. In the transferase family, the enzymeacyl-CoA:acetate-CoA transferase, also known as acetate-CoA transferase,is a suitable candidate to catalyze the desired 2-hydroxyisobutyryl-CoAor methacryl-CoA transferase activity due to its broad substratespecificity that includes branched acyl-CoA substrates (Matthies andSchink, Appl. Environ. Microbiol. 58:1435-1439 (1992); Vanderwinkel etal., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). ADP-formingacetyl-CoA synthetase (ACD) is a promising enzyme in the CoA synthetasefamily operating on structurally similar branched chain compounds(Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004); Musfeldt andSchonheit, J. Bacteriol. 184:636-644 (2002)). In the CoA-hydrolasefamily, the enzyme 3-hydroxyisobutyryl-CoA hydrolase has been shown tooperate on a variety of branched chain acyl-CoA substrates including3-hydroxyisobutyryl-CoA, methylmalonyl-CoA, and3-hydroxy-2-methylbutanoyl-CoA (Hawes et al., Methods Enzymol.324:218-228 (2000); Hawes et al., J. Biol. Chem. 271:26430-26434 (1996);Shimomura et al., J. Biol. Chem. 269:14248-14253 (1994)). Additionalexemplary gene candidates for CoA transferases, synthetases, andhydrolases are discussed in Example V (step 2 and 5).

Referring to FIG. 12, an alternative step 5 involves indirect conversionto MAA. As an alternative to direct conversion of MAA-CoA to MAA, analternate strategy for converting methacrylyl-CoA into MAA entails amulti-step process in which MAA-CoA is converted to MAA via3-hydroxyisobutyrate. By this process, MAA-CoA is first converted to3-hydroxyisobutyryl-CoA, which can subsequently be converted to MAA asdescribed in Example V.

The first step of this indirect route entails the conversion of MAA-CoAto 3-hydroxyisobutyryl-CoA (3HIB-CoA) by enoyl-CoA hydratase (EC4.2.1.17 and 4.2.1.74). In E. coli, the gene products of fadA and fadBencode a multienzyme complex involved in fatty acid oxidation thatexhibits enoyl-CoA hydratase activity (Nakahigashi and Inokuchi, NucleicAcids Research 18:4937 (1990); Yang, J. Bacteriol. 173:7405-7406 (1991);Yang et al., J. Biol. Chem. 265:10424-10429 (1990); Yang et al.,Biochemistry 30:6788-6795 (1991)). Knocking out a negative regulatorencoded by fadR can be utilized to activate the fadB gene product (Satoet al., J. Biosci. Bioengineer. 103:38-44 (2007)). The fadI and fadJgenes encode similar functions and are naturally expressed underanaerobic conditions (Campbell et al., Mol. Microbiol. 47:793-805(2003)).

Gene name GenBankID Organism fadA YP_026272.1 Escherichia coli fadBNP_418288.1 Escherichia coli fadI NP_416844.1 Escherichia coli fadJNP_416843.1 Escherichia coli fadR NP_415705.1 Escherichia coli

Additional native gene candidates encoding an enoyl-CoA hydrataseinclude maoC (Park and Lee, J. Bacteriol. 185:5391-5397 (2003)), paaF(Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003); Park and Lee,Appl. Biochem. Biotechnol. 113-116:335-346 (2004); Park and Yup,Biotechnol. Bioeng. 86:681-686. (2004)), and paaG (Ismail et al., Eur.J. Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem.Biotechnol. 113-116:335-346 (2004); Park and Yup, Biotechnol. Bioeng.86:681-686 (2004)). Non-native candidates include paaA, paaB, and paaNfrom P. putida (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424(1998)) and P. fluorescens (Di Gennaro et al., Arch. Microbiol.188:117-125 (2007)). The gene product of crt from C. acetobutylicum isanother candidate (Atsumi et al., Metab. Eng. epub Sep. 14, 2007;Boynton et al., J. Bacteriol. 178:3015-3024 (1996)).

Gene name GenBankID Organism maoC NP_415905.1 Escherichia coli paaFNP_415911.1 Escherichia coli paaG NP_415912.1 Escherichia coli paaANP_745427.1 Pseudomonas putida paaA ABF82233.1 Pseudomonas fluorescenspaaB NP_745426.1 Pseudomonas putida paaB ABF82234.1 Pseudomonasfluorescens paaN NP_745413.1 Pseudomonas putida paaN ABF82246.1Pseudomonas fluorescens crt NP_349318.1 Clostridium acetobutylicum

This example describes a biosynthetic pathway for production of2-hydroxyisobutyrate or MAA from acetyl-CoA.

Example XII Preparation of an MAA Producing Microbial Organism Having aPathway for Converting Acetyl-CoA to MAA

This example describes the generation of a microbial organism capable ofproducing MAA from acetyl-CoA.

Escherichia coli is used as a target organism to engineer the MAApathway shown in FIG. 12. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing MAA. E. coliis amenable to genetic manipulation and is known to be capable ofproducing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions.

To generate an E. coli strain engineered to produce MAA, nucleic acidsencoding the enzymes utilized in the pathway are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel supra, 1999). In particular, the atoB(P76461.1), hbd (P52041.2), and Mpe_B0541 (YP_(—)001023546.1) genesencoding the acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and3-hydroxybutyryl-CoA mutase activities, respectively, are cloned intothe pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. In addition, the hgdA (P11569), hgdB (P11570), hgdC (P11568),and hibch (Q5XIE6.2) genes encoding 2-hydroxyisobutyryl-CoA dehydrataseand methacrylyl-CoA hydrolase activities are cloned into the pZA33vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Thetwo sets of plasmids are transformed into E. coli strain MG1655 toexpress the proteins and enzymes required for MAA synthesis via theacetyl-CoA pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the MAA synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce MAAis confirmed using HPLC, gas chromatography-mass spectrometry (GCMS)and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MAA synthesis pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of MAA. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of MAA. Adaptiveevolution also can be used to generate better producers of, for example,the acetyl-CoA intermediate of the MAA product. Adaptive evolution isperformed to improve both growth and production characteristics (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science314:1565-1568 (2006)). Based on the results, subsequent rounds ofmodeling, genetic engineering and adaptive evolution can be applied tothe MAA producer to further increase production.

For large-scale production of MAA, the above organism is cultured in afermenter using a medium known in the art to support growth of theorganism under anaerobic conditions. Fermentations are performed ineither a batch, fed-batch or continuous manner Anaerobic conditions aremaintained by first sparging the medium with nitrogen and then sealingthe culture vessel, for example, flasks can be sealed with a septum andcrimp-cap. Microaerobic conditions also can be utilized by providing asmall hole in the septum for limited aeration. The pH of the medium ismaintained at a pH of around 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce MAA from acetyl-CoA.

Example XIII Pathway for Conversion of Acetyl-CoA to MAA viaCrotonoyl-CoA

This example describes an exemplary MAA synthetic pathway fromacetyl-CoA via crotonoyl-CoA.

Another route for converting acetyl-CoA to MAA in a minimum of sevenenzymatic steps is described (see FIG. 13). The yields of this pathwayunder aerobic and anaerobic conditions are similar to the pathwaydescribed in Example XI.

The first two steps of the pathway are identical to steps 1 and 2 in thepathway described in Example XI. In the third step, 3-HBCoA isdehydrated to form crotonyl-CoA by a crotonase (EC#: 4.2.1.55). Thedouble bond in crotonyl-CoA is reduced by butyryl-CoA dehydrogenase(EC#: 1.3.99.2). Both of these enzymes, just like the acetoacetyl-CoAreductase, are a part of the acetyl-CoA fermentation pathway to butyratein Clostridia species (Jones and Woods, Microbiol. Rev. 50:484-524(1986)). In the subsequent step, butyryl-CoA is converted intoisobutyryl-CoA by isobutyryl-CoA mutase (5.4.99.12), an enzyme that canreversibly convert butyryl-CoA into isobutyryl-CoA. This enzyme has beencloned and sequenced from Streptomyces cinnamonensis, and therecombinant enzyme has been characterized in E. coli (Ratnatilleke etal., J. Biol. Chem. 274:31679-31685 (1999)). The next step in thepathway entails the conversion of isobutyryl-CoA into methacrylyl-CoAvia 2-methyl-acylCoA dehydrogenase (EC #: 1.3.99.12). Thistransformation towards methacrylyl-CoA has been observed in Streptomycesspecies, and the associated enzyme has been isolated and expressed in E.coli (Youngleson et al., J. Bacteriol. 171:6800-6807 (1989)). In thefinal step, methacrylyl-CoA is converted to MAA by either a singleenzyme or a series of enzymes, as described in Example XI (step 5).

This example describes a biosynthetic pathway for production of MAA fromacetyl-CoA.

Example XIV Pathway for Conversion of Acrylyl-CoA to MAA

This example describes an exemplary MAA synthesis pathway fromacrylyl-CoA.

High yields of MAA can be obtained through the acrylyl-CoA pathway (seeFIG. 14). This pathway requires the activation of lactate to lactoyl-CoAfollowed by five, or optionally six, more steps for the conversion ofthis activated CoA molecule into MAA. The MAA yield from glucose usingthis pathway is 1.28 mol/mol of glucose and oxygen uptake is requiredfor attaining these yields. In the absence of oxygen, the expected yielddecreases from 1.28 mol to 1.09 mol/mol glucose consumed. Both theaerobic and anaerobic pathways are energy limited at maximum MAA yieldand do not generate any ATP.

MAA biosynthesis through the acrylyl-CoA pathway first requires theconversion of pyruvate into lactate via lactate dehydrogenase (EC1.1.1.28), an enzyme native to E. coli and many other organisms. Thethree subsequent steps, converting lactate into propionyl-CoA, arecatalyzed by enzymes in pyruvate fermentation pathways in severalunrelated bacteria such as Clostridium propionicum and Megasphaeraelsdenii (MetaCyc). Lactate-CoA transferase (EC 2.8.3.1), also known aspropionate-CoA transferase, converts lactate into lactoyl-CoA and canuse both propionate and lactate as substrates. This enzyme has beenpurified and characterized (Schweiger et al., Eur. J. Biochem.169:441-448 (1987)). Lactoyl-CoA is dehydrated into acrylyl-CoA usinglactoyl-CoA dehydratase (EC 4.2.1.54), an enzyme that has been a subjectof numerous studies (Hofineister and Buckel, Eur. J. Biochem.206:547-552. (1992); Kuchta and Abeles, J. Biol. Chem. 260:13181-13189(1985)). Subsequently, acrylyl-CoA is reduced to propionyl-CoA using theacryloyl-CoA reductase (EC 1.3.2.2, formerly 1.3.99.3) (Hetzel et al.,Eur. J Biochem. 270:902-910 (2003); Kuchta and Abeles, supra, 1985).

Referring to FIG. 14, in step 5, propionyl-CoA is converted intoS-methylmalonyl-CoA by propionyl-CoA carboxylase (6.4.1.3).Propionyl-CoA carboxylase has been purified from rat liver (Browner etal., J. Biol. Chem. 264:12680-12685 (1989); Kraus et al., J. Biol. Chem.258:7245-7248 (1983)) and has been isolated and characterized from humanliver as well (Kalousek et al., J. Biol. Chem. 255:60-65 (1980)).Carboxylation of propionyl-CoA into succinyl-CoA via this enzyme hasbeen identified as one of the mechanisms of propionate metabolism in E.coli (Evans et al., Biochem. J. 291 (Pt 3):927-932 (1993)), but verylittle is known about the genetics of the pathway.

The final steps of the pathway entail conversion of methylmalonyl-CoAinto MAA (lumped reaction in FIG. 14). Enzymes catalyzing thesereactions are described in Example I.

This example describes a biosynthetic pathway for production of MAA frompyruvate.

Example XV Pathway for Conversion of 2-Ketoisovalerate to MAA

This example describes an exemplary MAA synthetic pathway from2-ketoisovalerate

In this pathway, MMA biosynthesis occurs through 2-ketoisovalerate, aprecursor for valine biosynthesis (see FIG. 15). Specifically,2-ketoisovalerate can be formed from pyruvate following the action ofthree enzymes, acetolactate synthase, acetohydroxy acidisomeroreductase, and dihydroxy-acid dehydratase. The conversion of2-ketoisovalerate to MAA requires four enzymatic steps and leads to MAAyields of 1 mol/mol glucose under aerobic conditions and to yields of0.4 mol/mol glucose under anaerobic conditions (Table 5). The pathway isnot redox-balanced, and the secretion of fermentation products such asethanol and formate will occur under anaerobic conditions. In spite ofthe relatively low yields of MAA through this pathway in the absence ofoxygen, the energetics are very favorable and up to 2.2 moles of ATP aregenerated per mole of glucose consumed.

TABLE 5 Product and ATP yields for 2-ketoisovalerate pathway. MAAbiosynthetic pathway via 2- Ketoisovalerate Anaerobic Aerobic MAA Yield0.4 1.0 Max ATP yield @ max MAA yield 2.2 7.0

The pathway exploits multiple steps of the valine degradation routedescribed in several organisms, including Bacillus subtilis, Arabidopsisthaliana, and several species of Pseuodomonas but not known to bepresent in E. coli or in S. cerevisiae. In the first step of the valinedegradation pathway, valine is converted into 2-ketoisovalerate bybranched-chain amino acid aminotransferase (EC 2.6.1.24), an enzyme alsonative to E. coli (Matthies and Schink, Appl. Environ. Microbiol.58:1435-1439 (1992); Rudman and Meister, J. Biol. Chem. 200:591-604(1953)). The subsequent conversion of 2-ketoisovalerate intoisobutyryl-CoA, catalyzed by a branched-chain keto-acid dehydrogenasecomplex (EC 1.2.1.25), is the committing step for MAA biosynthesis viathis route. Next, isobutyryl-CoA is converted to methacrylyl-CoA viaisobutyryl-CoA dehydrogenase (EC 1.3.99.12). Details for this step aredescribed in Example XIII. The final step, conversion of MAA-CoA to MAA,is described in Example I.

This example describes a biosynthetic pathway for production of MMA from2-ketoisovalerate.

Example XVI Preparation of a 3-Hydroxyisobutyric Acid ProducingMicrobial Organism Having a Pathway for Converting 4-Hydroxybutyryl-CoAto 3-Hydroxyisobutyric Acid

This example describes the generation of a microbial organism capable ofproducing 3-hydroxyisobutyric acid from 4-hydroxybutyryl-CoA.

Escherichia coli is used as a target organism to engineer the3-hydroxyisobutyric acid pathway shown in FIG. 6. E. coli provides agood host for generating a non-naturally occurring microorganism capableof producing 3-hydroxyisobutyric acid. E. coli is amenable to geneticmanipulation and is known to be capable of producing various products,like ethanol, acetic acid, formic acid, lactic acid, and succinic acid,effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 3-hydroxyisobutyricacid, nucleic acids encoding the enzymes utilized in the pathway areexpressed in E. coli using well known molecular biology techniques (see,for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular,the sucD (YP_(—)001396394), 4hbd (YP_(—)001396393), buk1 (Q45829), andptb (NP_(—)349676) genes encoding succinic semialdehyde dehydrogenase(CoA-dependent), 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyratekinase, and phosphotransbutyrylase activities, respectively, are clonedinto the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. This construct allows the production of 4HB-CoA fromsuccinyl-CoA as described in Burk et al. (U.S. publication2009/0075351). In addition, the icmA (CAB40912.1) and hibch (Q5XIE6.2)genes encoding 4-hydroxybutyryl-CoA mutase and 3-hydroxyisobutyryl-CoAhydrolase, respectively, are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. The two sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for 3-hydroxyisobutyric acid synthesis viathe 4-hydroxybutyryl-CoA pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the 3-hydroxyisobutyric acid synthesis genes iscorroborated using methods well known in the art for determiningpolypeptide expression or enzymatic activity, including for example,Northern blots, PCR amplification of mRNA, immunoblotting, and the like.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individual activities. The ability of the engineered E.coli strain to produce 3-hydroxyisobutyric acid is confirmed using HPLC,gas chromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional 3-hydroxyisobutyricacid synthesis pathway are further augmented by optimization forefficient utilization of the pathway. Briefly, the engineered strain isassessed to determine whether any of the exogenous genes are expressedat a rate limiting level. Expression is increased for any enzymesexpressed at low levels that can limit the flux through the pathway by,for example, introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of 3-hydroxyisobutyric acid. Onemodeling method is the bilevel optimization approach, OptKnock (Burgardet al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied toselect gene knockouts that collectively result in better production of3-hydroxyisobutyric acid. Adaptive evolution also can be used togenerate better producers of, for example, the succinyl-CoA or4-hydroxybutyryl-CoA intermediates of the 3-hydroxyisobutyric acidproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the 3-hydroxyisobutyric acid producer tofurther increase production.

For large-scale production of 3-hydroxyisobutyric acid, the aboveorganism is cultured in a fermenter using a medium known in the art tosupport growth of the organism under anaerobic conditions. Fermentationsare performed in either a batch, fed-batch or continuous mannerAnaerobic conditions are maintained by first sparging the medium withnitrogen and then sealing the culture vessel, for example, flasks can besealed with a septum and crimp-cap. Microaerobic conditions also can beutilized by providing a small hole in the septum for limited aeration.The pH of the medium is maintained at a pH of around 7 by addition of anacid, such as H₂SO₄. The growth rate is determined by measuring opticaldensity using a spectrophotometer (600 nm) and the glucose uptake rateby monitoring carbon source depletion over time. Byproducts such asundesirable alcohols, organic acids, and residual glucose can bequantified by HPLC (Shimadzu, Columbia Md.), for example, using anAminex® series of HPLC columns (for example, HPX-87 series) (BioRad,Hercules Calif.), using a refractive index detector for glucose andalcohols, and a UV detector for organic acids (Lin et al., Biotechnol.Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce 3-hydroxyisobutyric acid from 4-hydroxybutyryl-CoA.

Example XVII Preparation of 2-Hydroxyisobutyric Acid Producing MicrobialOrganism Having a Pathway for Converting Acetyl-CoA to2-Hydroxyisobutyric Acid

This example describes the generation of a microbial organism capable ofproducing 2-hydroxyisobutyric acid from acetyl-CoA.

Escherichia coli is used as a target organism to engineer the2-hydroxyisobutyric acid pathway shown in FIG. 12. E. coli provides agood host for generating a non-naturally occurring microorganism capableof producing 2-hydroxyisobutyric acid. E. coli is amenable to geneticmanipulation and is known to be capable of producing various products,like ethanol, acetic acid, formic acid, lactic acid, and succinic acid,effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 2-hydroxyisobutyricacid, nucleic acids encoding the enzymes utilized in the pathway areexpressed in E. coli using well known molecular biology techniques (see,for example, Sambrook, supra, 2001; Ausubel, supra, 1999). Inparticular, the atoB (P76461.1), hbd (P52041.2), and Mpe_B0541(YP_(—)001023546.1) genes encoding the acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, and 3-hydroxybutyryl-CoA mutase activities,respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim,Germany) under the PA1/lacO promoter. In addition, hibch (Q5XIE6.2)encoding 2-hydroxyisobutyryl-CoA hydrolase activity is cloned into thepZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. The two sets of plasmids are transformed into E. coli strainMG1655 to express the proteins and enzymes required for2-hydroxyisobutyric acid synthesis via the acetyl-CoA pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the 2-hydroxyisobutyric acid synthesis genes iscorroborated using methods well known in the art for determiningpolypeptide expression or enzymatic activity, including for example,Northern blots, PCR amplification of mRNA, immunoblotting, and the like.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individual activities. The ability of the engineered E.coli strain to produce 2-hydroxyisobutyric acid is confirmed using HPLC,gas chromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional 2-hydroxyisobutyricacid synthesis pathway are further augmented by optimization forefficient utilization of the pathway. Briefly, the engineered strain isassessed to determine whether any of the exogenous genes are expressedat a rate limiting level. Expression is increased for any enzymesexpressed at low levels that can limit the flux through the pathway by,for example, introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of 2-hydroxyisobutyric acid. Onemodeling method is the bilevel optimization approach, OptKnock (Burgardet al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied toselect gene knockouts that collectively result in better production of2-hydroxyisobutyric acid. Adaptive evolution also can be used togenerate better producers of, for example, the acetyl-CoA intermediateof the 2-hydroxyisobutyric acid product. Adaptive evolution is performedto improve both growth and production characteristics (Fong and Palsson,Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568(2006)). Based on the results, subsequent rounds of modeling, geneticengineering and adaptive evolution can be applied to the2-hydroxyisobutyric acid producer to further increase production.

For large-scale production of 2-hydroxyisobutyric acid, the aboveorganism is cultured in a fermenter using a medium known in the art tosupport growth of the organism under anaerobic conditions. Fermentationsare performed in either a batch, fed-batch or continuous mannerAnaerobic conditions are maintained by first sparging the medium withnitrogen and then sealing the culture vessel, for example, flasks can besealed with a septum and crimp-cap. Microaerobic conditions also can beutilized by providing a small hole in the septum for limited aeration.The pH of the medium is maintained at a pH of around 7 by addition of anacid, such as H₂SO₄. The growth rate is determined by measuring opticaldensity using a spectrophotometer (600 nm) and the glucose uptake rateby monitoring carbon source depletion over time. Byproducts such asundesirable alcohols, organic acids, and residual glucose can bequantified by HPLC (Shimadzu, Columbia Md.), for example, using anAminex® series of HPLC columns (for example, HPX-87 series) (BioRad,Hercules Calif.), using a refractive index detector for glucose andalcohols, and a UV detector for organic acids (Lin et al., Biotechnol.Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce 2-hydroxyisobutyric acid from acetyl-CoA.

Example XVIII Pathway for Conversion of 4-hydroxybutyryl-CoA to2-Hydroxyisobutyrate or MAA via 2-Hydroxyisobutyryl-CoA

This example describes an exemplary 2-hydroxyisobutyrate or MAAsynthesis pathway proceeding from 4-hydroxybutyryl-CoA that passesthrough 2-hydroxyisobutyryl-CoA. The pathway, depicted in FIG. 12, ishigh-yielding under even under anaerobic conditions with a maximumtheoretical yield of 1.33 moles of 2-hydroxybutyrate or MAA per mole ofglucose. This is in contrast to the pathways originating from acetyl-CoAdescribed in Example XI, which are limited to a maximum theoreticalyield of one mole of product per mole of glucose.

The pathway first entails the dehydration of 4-hydroxybutyryl-CoA tovinylacetyl-CoA which is subsequently isomerized to crotonoyl-CoA.Crotonyl-CoA is hydrated to form 3-hydroxybutyryl-CoA, which isrearranged into 2-hydroxyisobutyryl-CoA. The final step of the2-hydroxyisobutyrate pathway involves eliminating the CoA functionalgroup from 2-hydroxyisobutyryl-CoA. The final steps in MAA synthesisinvolve the dehydration of 2-hydroxyisobutyryl-CoA followed by theremoval of the CoA functional group from methacrylyl-CoA. Genecandidates for the first three pathway steps, steps 7, 8, and 9 of FIG.12, are described below. Gene candidates for steps 3, 4, 5, and 6 ofFIG. 12 are discussed in example XI.

Referring to FIG. 12, steps 8 and 9 are carried out by4-hydroxybutyryl-CoA dehydratase enzymes. The enzymes from bothClostridium aminobutyrium and C. kluyveri catalyze the reversibleconversion of 4-hydroxybutyryl-CoA to crotonyl-CoA and also possess anintrinsic vinylacetyl-CoA Δ-isomerase activity (Scherf and Buckel, Eur.J. Biochem. 215:421-429 (1993); Scherf et al., Arch. Microbiol.161:239-245 (1994)). Both native enzymes have been purified andcharacterized, including the N-terminal amino acid sequences (Scherf andBuckel, supra, 1993; Scherf et al., supra, 1994). The abfD genes from C.aminobutyrium and C. kluyveri match exactly with these N-terminal aminoacid sequences, thus are encoding the 4-hydroxybutyryl-CoAdehydratases/vinylacetyl-CoA Δ-isomerase. In addition, abfD fromPorphyromonas gingivalis ATCC 33277 is another exemplary4-hydroxybutyryl-CoA dehydratase that can be identified throughhomology.

abfD YP_001396399.1 Clostridium kluyveri DSM 555 abfD P55792 Clostridiumaminobutyricum abfD YP_001928843 Porphyromonas gingivalis ATCC 33277

Step 10 of FIG. 12 is carried out by a crotonase enzyme. Such enzymesare required for n-butanol formation in some organisms, particularlyClostridial species, and also comprise one step of the3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilic Archaeaof the genera Sulfolobus, Acidianus, and Metallosphaera. Exemplary genesencoding crotonase enzymes can be found in C. acetobutylicum (Boynton,et al., J. Bacteriol. 178 (11):3015-3024 (1996)), C. kluyveri (Hillmerand Gottschalk, FEBS Lett. 21 (3):351-354 (1972)), and Metallosphaerasedula (Berg et al., Science 318 (5857):1782-1786 (2007)) though thesequence of the latter gene is not known. Enoyl-CoA hydratases, whichare involved in fatty acid beta-oxidation and/or the metabolism ofvarious amino acids, can also catalyze the hydration of crotonyl-CoA toform 3-hydroxybutyryl-CoA (Agnihotri and Liu, Bioorg. Med. Chem. 11(1):9-20 (2003); Roberts et al., Arch. Microbiol. 117 (1):99-108 (1978);Conrad et al., J. Bacteriol. 118 (1); 103-11 (1974)). The enoyl-CoAhydratases, phaA and phaB, of P. putida are believed to carry out thehydroxylation of double bonds during phenylacetate catabolism (Oliveraet al., Proc Natl Acad Sci USA 95 (11):6419-6424 (1998)). The paaA andpaaB from P. fluorescens catalyze analogous transformations (Olivera etal., supra, 1998). Lastly, a number of Escherichia coli genes have beenshown to demonstrate enoyl-CoA hydratase functionality including maoC(Park and Lee, J. Bacteriol. 185(18):5391-5397 (2003)), paaF (Park andLee, Biotechnol. Bioeng. 86(6):681-686 (2004a)); Park and Lee, Appl.Biochem. Biotechnol. 113-116: 335-346 (2004b)); Ismail et al. Eur. J.Biochem. 270(14):3047-3054 (2003), and paaG (Park and Lee, supra, 2004;Park and Lee, supra, 2004b; Ismail et al., supra, 2003).

crt NP_349318.1 Clostridium acetobutylicum crt1 YP_001393856 Clostridiumkluyveri DSM 555 paaA NP_745427.1 Pseudomonas putida paaB NP_745426.1Pseudomonas putida phaA ABF82233.1 Pseudomonas fluorescens phaBABF82234.1 Pseudomonas fluorescens maoC NP_415905.1 Escherichia colipaaF NP_415911.1 Escherichia coli paaG NP_415912.1 Escherichia coli

This example describes a biosynthesis pathway for 2-hydroxyisobutyrateor methacylic acid from 4-hydroxybutyryl-CoA.

Example XIX Preparation of an MAA Producing Microbial Organism Having aPathway for Converting 4-Hydroxybutyryl-CoA to MAA via2-Hydroxyisobutyryl-CoA

This example describes the generation of a microbial organism capable ofproducing MAA from 4-hydroxybutyryl-CoA via 2-hydroxyisobutyryl-CoA.

Escherichia coli is used as a target organism to engineer the MAApathway shown in FIG. 12 that starts from 4-hydroxybutyryl-CoA. E. coliprovides a good host for generating a non-naturally occurringmicroorganism capable of producing MAA. E. coli is amenable to geneticmanipulation and is known to be capable of producing various products,like ethanol, acetic acid, formic acid, lactic acid, and succinic acid,effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce MAA, nucleic acidsencoding the enzymes utilized in the pathway are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel, supra, 1999). First, the sucD(YP_(—)001396394), 4hbd (YP_(—)001396393), buk1 (Q45829), and ptb(NP_(—)349676) genes encoding succinic semialdehyde dehydrogenase(CoA-dependent), 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyratekinase, and phosphotransbutyrylase activities, respectively, are clonedinto the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. This construct allows the production of 4HB-CoA fromsuccinyl-CoA as described in Burk et al. (U.S. publication2009/0075351). The abfD (YP_(—)001396399.1) and crt1 (YP_(—)001393856)encoding 4-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ-isomerase,and enoyl-CoA hydratase activities, respectively, are cloned into thepZS23 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. In addition, the hgdA (P11569), hgdB (P11570), hgdC (P11568),and hibch (Q5XIE6.2) genes encoding 2-hydroxyisobutyryl-CoA dehydrataseand methacrylyl-CoA hydrolase activities are cloned into the pZS13vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter.pZS23 is obtained by replacing the ampicillin resistance module of thepZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistancemodule by well-known molecular biology techniques. The three sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for MAA synthesis from4-hydroxybutyryl-CoA via 2-hydroxyisobutyryl-CoA.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the MAA synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce MAAis confirmed using HPLC, gas chromatography-mass spectrometry (GCMS)and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional MAA synthesis pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of MAA. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of MAA. Adaptiveevolution also can be used to generate better producers of, for example,the 4-hydroxybutyryl-CoA intermediate of the MAA product. Adaptiveevolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the MAA producer to further increaseproduction.

For large-scale production of MAA, the above organism is cultured in afermenter using a medium known in the art to support growth of theorganism under anaerobic conditions. Fermentations are performed ineither a batch, fed-batch or continuous manner Anaerobic conditions aremaintained by first sparging the medium with nitrogen and then sealingthe culture vessel, for example, flasks can be sealed with a septum andcrimp-cap. Microaerobic conditions also can be utilized by providing asmall hole in the septum for limited aeration. The pH of the medium ismaintained at a pH of around 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce MAA from 4-hydroxybutyrl-CoA via 2-hydroxyisobutyryl-CoA.

Example XX Preparation of a 2-Hydroxyisobutyrate Producing MicrobialOrganism Having a Pathway for Converting 4-Hydroxybutyryl-CoA to2-Hydroxyisobutyrate via 2-Hydroxyisobutyryl-CoA

This example describes the generation of a microbial organism capable ofproducing 2-hydroxyisobutyrate from 4-hydroxybutyryl-CoA via2-hydroxyisobutyryl-CoA.

Escherichia coli is used as a target organism to engineer the2-hydroxyisobutyrate pathway shown in FIG. 12 that starts from4-hydroxybutyryl-CoA. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing2-hydroxyisobutyrate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce2-hydroxyisobutyrate, nucleic acids encoding the enzymes utilized in thepathway are expressed in E. coli using well known molecular biologytechniques (see, for example, Sambrook, supra, 2001; Ausubel supra,1999). First, the sucD (YP_(—)001396394), 4hbd (YP_(—)001396393), buk1(Q45829), and ptb (NP_(—)349676) genes encoding succinic semialdehydedehydrogenase (CoA-dependent), 4-hydroxybutyrate dehydrogenase,4-hydroxybutyrate kinase, and phosphotransbutyrylase activities,respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim,Germany) under the PA1/lacO promoter. This construct allows theproduction of 4HB-CoA from succinyl-CoA as described in Burk et al.(U.S. publication 2009/0075351). The abfD (YP_(—)001396399.1), crt1(YP_(—)001393856), and hibch (Q5XIE6.2) genes encoding4-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ-isomerase, enoyl-CoAhydratase, and 2-hydroxyisobutyryl-CoA hydrolase activities,respectively, are cloned into the pZS23 vector (Expressys, Ruelzheim,Germany) under the PA1/lacO promoter. The two sets of plasmids aretransformed into E. coli strain MG1655 to express the proteins andenzymes required for 2-hydroxyisobutyrate synthesis from4-hydroxybutyryl-CoA via 2-hydroxyisobutyryl-CoA.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). Cobalamin is alsosupplied to the medium to ensure activity of the mutase enzyme unlessthe host strain of E. coli is engineered to synthesize cobalamin de novo(see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). Theexpression of the 2-hydroxyisobutyrate synthesis genes is corroboratedusing methods well known in the art for determining polypeptideexpression or enzymatic activity, including for example, Northern blots,PCR amplification of mRNA, immunoblotting, and the like. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individual activities. The ability of the engineered E. colistrain to produce 2-hydroxyisobutyrate is confirmed using HPLC, gaschromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional 2-hydroxyisobutyratesynthesis pathway are further augmented by optimization for efficientutilization of the pathway. Briefly, the engineered strain is assessedto determine whether any of the exogenous genes are expressed at a ratelimiting level. Expression is increased for any enzymes expressed at lowlevels that can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of 2-hydroxyisobutyrate. One modelingmethod is the bilevel optimization approach, OptKnock (Burgard et al.,Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to selectgene knockouts that collectively result in better production of2-hydroxyisobutyrate. Adaptive evolution also can be used to generatebetter producers of, for example, the 4-hydroxybutyryl-CoA intermediateof the 2-hydroxyisobutyrate product. Adaptive evolution is performed toimprove both growth and production characteristics (Fong and Palsson,Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568(2006)). Based on the results, subsequent rounds of modeling, geneticengineering and adaptive evolution can be applied to the2-hydroxyisobutyrate producer to further increase production.

For large-scale production of 2-hydroxyisobutyrate, the above organismis cultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing the culture vessel, for example, flasks can be sealed witha septum and crimp-cap. Microaerobic conditions also can be utilized byproviding a small hole in the septum for limited aeration. The pH of themedium is maintained at a pH of around 7 by addition of an acid, such asH₂SO₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of a microbial organism that canproduce 2-hydroxyisobutyrate from 4-hydroxybutyrl-CoA via2-hydroxyisobutyryl-CoA.

Example XXI Design of Gene Knockout Strains for Increased Production ofMethacrylic Acid or 3-Hydroxyisobutyric Acid

This example describes the design of strains with gene knockouts forincreased production of methacrylic acid or 3-hydroxyisobutyric acid.

OptKnock is a bilevel computational framework formulated with theoverall objective of developing genetically stable overproducingmicroorganisms. Specifically, the framework examines the completenetwork of a microorganism in order to suggest genetic manipulationsthat force the desired biochemical to become an obligatory byproduct ofcell growth. By coupling biochemical production with cell growth throughstrategically placed gene deletions, the growth selection pressuresimposed on the engineered strains after long periods of time in abioreactor lead to improvements in performance as a result of thecompulsory growth-coupled biochemical production. Lastly, there isnegligible possibility of the designed strains reverting to theirwild-type states because the genes selected by OptKnock are to becompletely removed from the genome by appropriate genetic manipulationsusing a complete deletion rather than an insertion.

The concept of growth-coupled biochemical production can be visualizedin the context of the biochemical production envelopes of a typicalmetabolic network calculated using an in silico model. These limits areobtained by fixing the uptake rate(s) of the limiting substrate(s) totheir experimentally measured value(s) and calculating the maximum andminimum rates of biochemical production at each attainable level ofgrowth. Although exceptions exist, typically the production of a desiredbiochemical is in direct competition with biomass formation forintracellular resources (see FIG. 16). Thus, enhanced rates ofbiochemical production will necessarily result in sub-maximal growthrates. The knockouts suggested by OptKnock are designed to restrict theallowable solution boundaries, forcing a change in metabolic behaviorfrom the wild-type strain. Although the actual solution boundaries for agiven strain will expand or contract as the substrate uptake rate(s)increase or decrease, each experimental point should lie within itscalculated solution boundary. Plots such as these allow thevisualization of how close strains are to their performance limits or,in other words, how much room is available for improvement. The OptKnockframework has already been able to identify promising gene deletionstrategies for biochemical overproduction (Burgard et al., Biotechnol.Bioeng. 84 (6):647-657 (2003); Pharkya et al., Biotechnol. Bioeng. 84(7):887-899 (2003); Pharkya et al., Genome Res. 14 (11):2367-2376(2004)) and establishes a systematic framework that will naturallyencompass future improvements in metabolic and regulatory modelingframeworks.

Described in more detail in Examples XXII and XXIII are sets of enzymeactivities that should be absent, attenuated, or eliminated for creatinghost organisms that achieve growth-coupled MAA or 3-hydroxyisobutyricacid production upon the addition of the MAA or 3-hydroxyisobutyric acidbiosynthetic pathways. To enumerate all potential strategies, anoptimization technique, termed integer cuts, has been implemented whichentails iteratively solving the OptKnock problem with the incorporationof an additional constraint referred to as an integer cut at eachiteration.

The OptKnock algorithm identified growth-coupled strain designs foroverproduction of MAA, or if desired the precursor 3-hydroxyisobutyrate(3-HIB), based on a stoichiometric model of Escherichia coli metabolism.Assumptions include (i) a glucose uptake rate of 10 mmol/gdw/hr; (ii)anaerobic or microaerobic conditions; and (iii) a minimum non-growthassociated maintenance requirement of 4 mmol/gDCW/hr. Although thegrowth substrate was assumed to be glucose, it is understood that thestrategies are applicable to any substrate including glucose, sucrose,xylose, arabinose, or glycerol. The complete set of growth-coupledproduction designs for the succinyl-CoA:MAA pathway (FIG. 2) and the4-HB-CoA:MAA pathway (FIG. 6) are listed in Tables 10 and 11,respectively. Tables 10 and 11 show the reaction combinations targetedfor removal by OptKnock to enhance production of MAA or3-hydroxyisobutyric acid via a succinyl-CoA (Table 10) or4-hydroxybutyryl-CoA (Table 11) intermediate. Attenuation of at leastone, or any combination of the reactions, including up to most or all ofthe reactions, can be utilized to achieve a desired effect. The enzymenames, their abbreviations, and the corresponding reactionstoichiometries are listed in Table 12. Finally, metabolite namescorresponding to the abbreviations in the reaction equations are listedin Table 13.

Although the designs were identified using a metabolic model of E. colimetabolism, and the gene names listed in Table 12 are specific to E.coli, the method of choosing the metabolic engineering strategies andalso the designs themselves are applicable to any MAA or3-hydroxyisobutyrate producing organism. Thus the designs areessentially lists of enzymatic transformations whose activity must beeither eliminated, attenuated, or initially absent from a microorganismto enable growth coupled production.

The key criterion for prioritizing the final selection of designs wasthe growth-coupled yield of 3-hydroxyisobutyrate and/or methacrylicacid. To examine this, production cones were constructed for eachstrategy by first maximizing and subsequently minimizing product yieldsat different rates of biomass formation, as described above. Convergenceof the rightmost boundary of all possible phenotypes of the mutantnetwork at a single point implies that there is a unique optimum yieldof the product at the maximum biomass formation rate. In other cases,the rightmost boundary of the feasible phenotypes is a vertical line,indicating that at the point of maximum biomass, the network can makeany amount of MAA in the calculated range, including the lowest amountat the bottommost point of the vertical line. Such designs were given alower priority. Short lists of the highest priority OptKnock designs foreach pathway are provided in Tables 6 and 8 in Examples XXII and XXIII,respectively.

Although strain designs in the following Examples are characterized bytheir capacity to produce MAA coupled to biomass formation, it isunderstood that these strains can also be utilized to overproduce theMAA-pathway intermediate 3-hydroxyisobutyrate. In both pathways, thefinal enzymatic step for forming MAA entails the dehydration of3-hydroxyisobutyrate by 3-hydroxyisobutyrate dehydratase (step 5 in FIG.2, step 3 in FIG. 6). Since this reaction does not consume or producereducing equivalents, protons, or energy it will not alter theenergetics of the strain designs. Thus, in a strain lacking3-hydroxyisobutyrate dehydratase activity, all designs described hereinallow growth-coupled production of 3-HIB.

This example describes the design of gene knockouts for generatingstrains for growth coupled production of MAA and/or 3-HIB.

Example XXII Knockout Designs for a Succinyl-CoA:MAA Pathway

This example describes knockout designs for a succinyl-CoA to MAApathway. As discussed previously, it is understood that similar knockoutdesigns can be used for a succinyl-CoA to 3-hydroxyisobutyrate pathwayas well.

Table 6 shows growth coupled designs for the succinyl-CoA to MAApathway, designed as described in Example XXI. Table 7 shows maximumtheoretical yields of MAA and biomass formation rates of growth-coupleddesigns shown in Table 6.

TABLE 6 Sets of enzymatic transformations whose activity should beeither eliminated, attenuated or initially absent from a microorganismto allow the growth coupled production of methyacrylic acid and/or3-hydroxyisobutyrate. Design Enzyme activity Abbreviation Notes 1Acetaldehyde-CoA dehydrogenase ADHEr malate dehydrogenase MDH D-lactatedehydrogenase LDH_D 2 Acetaldehyde-CoA dehydrogenase ADHEr Design 1 +ASPT malate dehydrogenase MDH L-aspartase ASPT D-lactate dehydrogenaseLDH_D 3 Acetaldehyde-CoA dehydrogenase ADHEr Design 2 + PFLi malatedehydrogenase MDH L-aspartase ASPT D-lactate dehydrogenase LDH_Dpyruvate formate lyase PFLi 4 Acetaldehyde-CoA dehydrogenase ADHErDesign 3 + THD2 and/or GLUDy malate dehydrogenase MDH L-aspartase ASPTD-lactate dehydrogenase LDH_D pyruvate formate lyase PFLi NAD(P)transhydrogenase THD2 and/or and/or glutamate dehydrogenase (NADP) GLUDy5 Acetaldehyde-CoA dehydrogenase ADHEr Design 2 + ATPS4r malatedehydrogenase MDH L-aspartase ASPT D-lactate dehydrogenase LDH_D ATPsynthase ATPS4r 6 Acetaldehyde-CoA dehydrogenase ADHEr Design 5 + GLCptsmalate dehydrogenase MDH L-aspartase ASPT D-lactate dehydrogenase LDH_DATP synthase ATPS4r D-glucose transport via PEP:Pyr PTS GLCpts 7Acetaldehyde-CoA dehydrogenase ADHEr Design 1 + GLUDy malatedehydrogenase MDH D-lactate dehydrogenase LDH_D glutamate dehydrogenase(NADP) GLUDy 8 Acetaldehyde-CoA dehydrogenase ADHEr Design 7 + PFLimalate dehydrogenase MDH D-lactate dehydrogenase LDH_D glutamatedehydrogenase (NADP) GLUDy pyruvate formate lyase PFLi 9Acetaldehyde-CoA dehydrogenase ADHEr Design 8 + ACKr and/or PTAr malatedehydrogenase MDH D-lactate dehydrogenase LDH_D pyruvate formate lyasePFLi glutamate dehydrogenase (NADP) GLUDy Phosphotransacetylase and/oracetate kinase ACKr and/or PTAr 10 Acetaldehyde-CoA dehydrogenase ADHErDesign 1 + THD2 malate dehydrogenase MDH D-lactate dehydrogenase LDH_DNAD(P) transhydrogenase THD2 11 Acetaldehyde-CoA dehydrogenase ADHErDesign 10 + PGL and/or G6PDHy malate dehydrogenase MDH D-lactatedehydrogenase LDH_D NAD(P) transhydrogenase THD26-phosphogluconolactonase and/or PGL and/or glucose 6-phosphatedehydrogenase G6PDHy 12 Acetaldehyde-CoA dehydrogenase ADHEr Design 11 +PFLi malate dehydrogenase MDH D-lactate dehydrogenase LDH_D NAD(P)transhydrogenase THD2 6-phosphogluconolactonase and/or PGL and/orglucose 6-phosphate dehydrogenase G6PDHy pyruvate formate lyase PFLi 13Acetaldehyde-CoA dehydrogenase ADHEr Design 1 + NADH6 malatedehydrogenase MDH D-lactate dehydrogenase LDH_D NADH dehydrogenase NADH614 Acetaldehyde-CoA dehydrogenase ADHEr Design 13 + ACKr/PTAr malatedehydrogenase MDH D-lactate dehydrogenase LDH_D NADH dehydrogenase NADH6Phosphotransacetylase and/or acetate kinase ACKr and/or PTAr

All high-priority growth coupled designs for the succinyl-CoA to MAApathway (Table 6 and FIG. 17) build upon Design 1, which calls for theabsence of acetylaldehyde-CoA dehydrogenase (ADHEr), malatedehydrogenase (MDH), and lactate dehydrogenase (LDH_D) activities toprevent the formation of fermentation byproducts. Design 2 builds uponthis base design with the additional removal of L-aspartase (ASPT)functionality. This design is capable of reaching 54% of the theoreticalmaximum MAA yield (0.35 g/g) at the maximum biomass yield (Table 7).

TABLE 7 Maximum theoretical MAA yields and biomass formation rates ofgrowth-coupled designs in Table 6. The maximum theoretical yield of MAAin a wild-type background is 0.64 g/g (grams MAA produced per gramglucose utilized). Design MAA (g/g) % Theoretical Yield Biomass (1/hr) 20.35 54% 0.148 3 0.60 94% 0.076 4 0.60 95% 0.07 5 0.42 66% 0.123 6 0.5484% 0.07 7 0.21 32% 0.162 8 0.35 55% 0.123 9 0.60 95% 0.07 10 0.29 46%0.202 11 0.37 59% 0.158 12 0.52 82% 0.131 13 0.27 42% 0.195 14 0.56 88%0.118

Designs 3 and 4 build on Design 2 as a base design. Design 3 entails theremoval of pyruvate formate lyase (PFLi) activity to prevent secretionof formate as a byproduct. This design results in an MAA yield of 94% ofthe theoretical maximum. Further deletion of NAD(P) transhydrogenase(THD2) and/or glutamate dehydrogenase (GLUDy) in Design 4 serves totightly couple cell growth to MAA production while achieving 95% of thetheoretical maximum yield. This design also requires the formation of atleast 0.24 g/g MAA for biomass formation.

Designs 5 and 6 also build on Design 2 as a base design. In Design 5,removal of ATP synthase (ATPS4r) results in a yield of 0.42 g/g MAA atthe maximum biomass formation rate of 0.123 l/hr. This design tightlycouples growth to product formation but requires secretion of acetateand formate as fermentation byproducts. Removing glucose transport viathe phosphoenolpyruvate:pyruvate PTS system reduces byproduct formationand increases MAA production to 0.54 g/g (84% of the maximum theoreticalyield).

Designs 7-14 build on Design 1, in which ADHEr, MDH and LDH_Dfunctionality is removed. In Design 7, removal of glutamatedehydrogenase (GLUDy) functionality yields a mutant that produces 0.21g/g MAA at 0.162 l/hr. Further deletion of pyruvate formate lyase (PFLi)in Design 8 yields 0.35 g/g MAA. Additional deletion ofphosphotransacetylase (PTAr) and/or acetate kinase (ACKr) in Design 9prevents formation of acetate and increases product yield to 0.60 g/g,95% of the theoretical maximum. Further removal of transhydrogenase(THD2) functionality improves growth-coupling of this design.

Design 10 knocks out NAD(P) transhydrogenase (THD2) in addition to MDH,LDH, and ADHEr. This strain is predicted to achieve an MAA yield of 0.29g/g at a maximum growth rate of 0.20 l/hr. Additional deletion of6-phosphogluconolactonase (PGL) and/or glucose-6-phosphate dehydrogenase(G6PDHy) serves to increase flux through glycolysis, thereby improvingthe predicted MAA yield to 0.37 g/g with tightened coupling to biomassformation. Additional deletion of pyruvate formate lyase (PFLi), whichforces flux through PDH and reduces byproduct formation, increases thepredicted MAA yield to 0.52 g/g, 82% of the theoretical maximum.Additional deletions in ACKr and ASPT also improve the product yield ofthis design by reducing byproduct formation.

Design 13 builds on Design 1 with the additional knockout of NADHdehydrogenase (NADH6). This yields a strain with an MAA yield of 0.27g/g at the maximum biomass formation rate. Further deletion ofphosphotransacetylase (PTAr) and/or acetate kinase (ACKr) in Design 14improves the yield to 0.56 g/g, 88% of the theoretical maximum. Thisdesign has the advantage of producing MAA as the sole fermentationbyproduct.

All high-yielding strain designs involve deletion of at least one of thefollowing reactions: alcohol dehydrogenase (ADHEr), malate dehydrogenase(MDH), lactate dehydrogenase (LDH_D), phosphogluconolactonase (PGL),glucose-6-phosphate dehydrogenase (G6PDHy), pyruvate formate lyase(PFLi), NAD(P) transhydrogenase (THD2), ATP synthetase (ATPS4r),glutamate dehydrogenase (GLUDy), aspartase (ASPT), acetate kinase(ACKr), phosphotransacetylase (PTAr) and NADH dehydrogenase (NADH6).Addition of any of these knockouts to the strain designs in Table 6 willfurther improve the yield of MAA or 3-hydroxyisobutryrate.

These results describe knockout design strategies to generate strainshaving growth-coupled production of MAA or 3-HIB.

Example XXIII Knockout Designs for a 4-Hydroxybutyryl-CoA:MAA Pathway

This example describes knockout designs for a 4-hydroxybutyryl-CoA toMAA pathway. As discussed previously, it is understood that similarknockout designs can be used for a 4-hydroxybutyryl-CoA to3-hydroxyisobutyrate pathway as well.

For the 4-hydroxybutyryl-CoA pathway, OptKnock designs were generatedfor strains that utilize either a hydrolase or a transferase to generate3-hydroxyisobutyrate or MAA (FIG. 6, step 2). Designs generated for thetwo conditions were similar, although product yields and growth-couplingwere significantly higher when a transferase is utilized. All designsare listed in Table 11. Table 8 shows growth coupled designs for the4-hydroxybutyryl-CoA to MAA pathway, designed as described in ExampleXXI. Table 9 shows maximum theoretical yields of MAA and biomassformation rates of growth-coupled designs shown in Table 8.

TABLE 8 Sets of enzymatic transformations whose activity should beeither eliminated, attenuated or initially absent from a microorganismto allow the growth coupled production of methacrylic acid and/or3-hydroxyisobutyric acid. Design Enzyme activity Abbreviation Notes 1Acetaldehyde-CoA dehydrogenase ADHEr malate dehydrogenase MDH D-lactatedehydrogenase LDH_D 2 Acetaldehyde-CoA dehydrogenase ADHEr Design 1 +ASPT malate dehydrogenase MDH D-lactate dehydrogenase LDH_D L-aspartaseASPT 3 Acetaldehyde-CoA dehydrogenase ADHEr Design 2 + THD2/GLUDy malatedehydrogenase MDH D-lactate dehydrogenase LDH_D L-aspartase ASPT NAD(P)transhydrogenase THD2 and/or and/or glutamate dehydrogenase (NADP) GLUDy4 Acetaldehyde-CoA dehydrogenase ADHEr Design 3 + PFLi malatedehydrogenase MDH L-aspartase ASPT D-lactate dehydrogenase LDH_D NAD(P)transhydrogenase THD2 and/or and/or glutamate dehydrogenase (NADP) GLUDypyruvate formate lyase PFLi 5 Acetaldehyde-CoA dehydrogenase ADHErDesign 2 + ATPS4r malate dehydrogenase MDH D-lactate dehydrogenase LDH_DL-aspartase ASPT ATP synthase ATPS4r 6 Acetaldehyde-CoA dehydrogenaseADHEr Design 5 + PGL and/or G6PDHy malate dehydrogenase MDH D-lactatedehydrogenase LDH_D L-aspartase ASPT ATP synthase ATPS4r6-phosphogluconolactonase and/or PGL and/or glucose 6-phosphatedehydrogenase G6PDHy 7 Acetaldehyde-CoA dehydrogenase ADHEr Design 5 +PFLi malate dehydrogenase MDH D-lactate dehydrogenase LDH_D L-aspartaseASPT ATP synthase ATPS4r Pyruvate formate lyase PFLi 8 Acetaldehyde-CoAdehydrogenase ADHEr Design 1 + THD2 malate dehydrogenase MDH D-lactatedehydrogenase LDH_D NAD(P) transhydrogenase THD2 and/or and/or glutamatedehydrogenase (NADP) GLUDy 9 Acetaldehyde-CoA dehydrogenase ADHEr Design8 + PGL and/or G6PDHy malate dehydrogenase MDH D-lactate dehydrogenaseLDH_D NAD(P) transhydrogenase THD2 and/or and/or glutamate dehydrogenase(NADP) GLUDy 6-phosphogluconolactonase and/or PGL and/or glucose6-phosphate dehydrogenase G6PDHy

The highest priority growth-coupled strain designs (Table 8, FIG. 18)build upon Design 1, a base strain with removed, reduced or attenuatedalcohol dehydrogenase (ADHEr), malate dehydrogenase (MDH) and lactatedehydrogenase (LDH_D) functionality. The additional removal of succinatesemialdehyde dehydrogenase functionality may be beneficial for efficientchanneling of flux through succinyl-CoA.

TABLE 9 Maximum theoretical MAA yields and biomass formation rates ofgrowth-coupled designs in Table 8. MAA yields are calculated under theassumption that a transferase is utilized to convert 3-hydroxyisobutyrl-CoA to 3-hydroxyisobutyrate. Identical yields are predicted if3-hydroxyisobutyryl-CoA is first converted to methacryl-CoA which isthen converted to MAA by a transferase. Additional assumptions: ATPmaintenance energy = 4 mmol/gDCW/hr, SSALx, SSALy knocked out. DesignMAA Yield (g/g) % Theoretical Max Biomass (1/hr) 2 0.33 52% 0.13 3 0.3454% 0.12 4 0.62 97% 0.039 5 0.31 48% 0.073 6 0.57 90% 0.056 7 0.56 88%0.044 8 0.29 46% 0.19 9 0.52 81% 0.112

Designs 2-7 build on Design 1 with the additional removal, reduction orattenuation of L-aspartase (ASPT) activity. Design 2 produces an MAAyield of 0.33 g/g at the maximum growth rate of 0.13 l/hr. The majorfermentation byproducts of this strain are acetate and formate. Furtherdeletion of genes involved in formate production and energy generationcan reduce formation of these byproducts. Design 3 builds upon Design 2with the additional deletion of NAD(P) transhydrogenase (THD2) and/orglutamate dehydrogenase (GLUDy), resulting in an MAA yield of 0.34 g/gat the maximum growth rate 0.12 l/hr. This strain does not eliminatebyproduct formation, but it is tightly growth-coupled and is required toproduce a minimum of 0.07 g MAA per gram glucose utilized for energygeneration. Additional deletion of pyruvate formate lyase (PFLi) inDesign 4 eliminates formate secretion and increases the MAA yield to0.62 g/g (97% of the theoretical maximum) and also requires theproduction of at least 0.24 g/g MAA for energy generation.

Design 5 builds upon Design 4 with the additional deletion of ATPsynthetase (ATPS4r). This strain achieves 0.31 g/g MAA at a maximumgrowth rate of 0.073 l/hr. Further deletion of 6-phosphogluconolactonase(PGL) and/or glucose-6-phosphate dehydrogenase (G6PDHy) functionality inDesign 6 increases the product yield at maximum biomass (0.57 g/g at0.056 l/hr maximum growth rate). Alternatively, deletion of pyruvateformate lyase also results in a high-yielding design with tightgrowth-coupling (Design 7).

Design 8 builds upon the Design 1 base strain (ADHEr, LDH_D, MDH) withthe removal of NAD(P) transhydrogenase functionality. This strain designachieves 0.29 g/g MAA at biomass 0.19 l/hr. Further deletion ofphosphogluconolactonase (PGL) and/or glucose-6-phosphate dehydrogenase(G6PDHy) in Design 9 increases MAA production at maximum biomass to 0.52g/g at 0.112 l/hr.

All high-yielding strain designs involve deletion of at least one of thefollowing reactions: alcohol dehydrogenase (ADHEr), malate dehydrogenase(MDH), lactate dehydrogenase (LDH_D), phosphogluconolactonase (PGL),glucose-6-phosphate dehydrogenase (G6PDHy), pyruvate formate lyase(PFLi), NAD(P) transhydrogenase (THD2), ATP synthetase (ATPS4r),glutamate dehydrogenase (GLUDy), and aspartase (ASPT). Addition of anyof these knockouts to the strain designs in Table 8 will further improvethe yield of MAA or 3-hydroxyisobutryrate.

These results describe knockout design strategies to generate strainshaving growth-coupled production of MAA or 3-HIB.

Example XXIV Characterization of Engineered Strains

This example describes characterization of engineered strains.

Strain construction: Escherichia coli K-12 MG1655 housing the3-hydroxyisobutyrate and/or MAA pathway is used as the strain into whichthe deletions are introduced. The strains are constructed byincorporating in-frame deletions using homologous recombination via theλ Red recombinase system of Datsenko and Wanner (Proc. Natl. Acad. Sci.USA 97 (12):6640-6645 2000)). The approach involves replacing achromosomal sequence, that is, the gene targeted for removal, with aselectable antibiotic resistance gene, which itself is later removed.The knockouts are integrated one by one into the recipient strain. Noantibiotic resistance markers remain after each deletion, allowingaccumulation of multiple mutations in each target strain. The deletiontechnology completely removes the gene targeted for removal so as tosubstantially reduce the possibility of the constructed mutantsreverting back to the wild-type.

Shake flask characterization: As intermediate strains are constructed,strain performance is quantified by performing shake flaskfermentations. Anaerobic conditions are obtained by sealing the flaskswith a rubber septum and then sparging the medium with nitrogen. Forstrains where growth is not observed under strict anaerobic conditions,microaerobic conditions are applied by covering the flask with foil andpoking a small hole for limited aeration. Experiments are performedusing M9 minimal medium supplemented with glucose unless otherwisedesired for a particular application. Pre-cultures are grown overnightand used as inoculum for a fresh batch culture for which measurementsare taken during exponential growth. The growth rate is determined bymeasuring optical density using a spectrophotometer (600 nm), and theglucose uptake rate by monitoring carbon source depletion over time.Ethanol, MAA, 3-hydroxyisobutyric acid and organic acids are analyzed byGC-MS or HPLC using routine procedures. Triplicate cultures are grownfor each strain.

Batch Fermenter Testing: The performance of selected strains are testedin anaerobic, pH-controlled batch fermentations. This allows reliablequantification of the growth, glucose uptake, and formation rates of allproducts, as well as ensure that the accumulation of acidic fermentationproducts will not limit cell growth. In addition, it allows accuratedetermination of 3-hydryxoyisobutyric acid and/or MAA volumetricproductivity and yield, two of the most important parameters inbenchmarking strain performance. Fermentations are carried out in 1-Lbioreactors with 600 mL working volume, equipped with temperature and pHcontrol. The reactor is continuously sparged with N₂ at approximately0.5 L/min to ensure that dissolved oxygen (DO) levels remain belowdetection levels. The culture medium is the same as described above,except that the glucose concentration is increased in accordance withthe higher cell density achievable in a fermentation vessel.

Chemostat Testing: Chemostat experiments are conducted to obtain adirect measure of how the switch in fermentation mode from batch tocontinuous affects 3-hydroxyisobutyric acid and/or MAA yield andvolumetric productivity. The bioreactors described above using batchmode are operated in chemostat mode through continuous supply of mediumand removal of spent culture. The inlet flow rate is set to maintain aconstant dilution rate of 80% of the maximum growth rate observed foreach strain in batch, and the outlet flow is controlled to maintainlevel. Glucose is the limiting nutrient in the medium and is set toachieve the desired optical density in the vessel.

Adaptive evolution: The knockout strains are expected initially toexhibit suboptimal growth rates until their metabolic networks haveadjusted to their missing functionalities. To facilitate thisadjustment, the strains are adaptively evolved. By subjecting thestrains to adaptive evolution, cellular growth rate becomes the primaryselection pressure and the mutant cells are compelled to reallocatetheir metabolic fluxes in order to enhance their rates of growth. Thisreprogramming of metabolism has been recently demonstrated for severalE. coli mutants that had been adaptively evolved on various substratesto reach the growth rates predicted a priori by an in silico model (Fongand Palsson, Nat. Genet. 36(10):1056-1058 (2004)). TheOptKnock-generated strains are adaptively evolved in triplicate (runningin parallel) due to differences in the evolutionary patterns witnessedpreviously in E. coli (Fong and Palsson, Nat. Genet. 36(10):1056-1058(2004); Fong et al., J. Bacteriol. 185(21):6400-6408 (2003); Ibarra etal., Nature 420(6912):186-189 (2002)) that could potentially result inone strain having superior production qualities over the others.Evolutions are run for a period of 2-6 weeks, depending upon the rate ofgrowth improvement attained. In general, evolutions are stopped once astable phenotype is obtained. The growth-coupled biochemical productionconcept behind the OptKnock approach results in the generation ofgenetically stable overproducers.

As described above and in previous examples, strain engineeringstrategies for coupling methacrylic acid (MAA) and 3-hydroxyisobutyrate(3-HIB) production to cell growth were calculated using OptKnockmethodology. Two pathways were explored. The first pathway proceedsthrough methylmalonyl-CoA as an intermediate. The second pathwayproceeds through 4-hydroxybutyryl-CoA and can utilize either a CoAtransferase, hydrolase or synthetase to convert 3-hydroxyisobutyryl-CoAto 3-HIB. Alternatively, MAA can be produced directly by this pathway if3-hydroxyisobutyryl-CoA is first converted to methacrylyl-CoA. Pathwayselection, host background, and selection of enzymes for each particularstep impact product yield and growth characteristics of the finalproduction strain.

Assuming that 3-hydroxyisobutyric acid is produced as a precursor, thefinal step of both pathways entails dehydration of 3-HIB to MAA by3-hydroxyisobutyrate dehydratase. As this conversion does not requireenergy or redox equivalents, it is understood that the strain designstrategies described for MAA can also be applied for growth-coupledproduction of 3-HIB production if 3-hydroxyisobutyrate dehydrataseactivity is not present in the production organism. In this case, thenon-naturally occurring organism would produce 3-HIB instead of MAA. Themaximum theoretical product and energetic yields are unchangedregardless of whether MAA or 3-HIB is produced.

All high-priority strain designs are built on three central deletions:MDH, LDH_D and ADHEr. This analysis revealed that host strain designstrategies are remarkably similar and involve the deletion of a smallnumber of enzyme activities in the host organism. The main enzymeactivities impacting MAA (or 3-HIB) production are: acetaldehyde-CoAdehydrogenase (ADHEr), malate dehydrogenase (MDH), lactate dehydrogenase(LDH_D), phosphogluconolactonase (PGL), glucose-6-phosphatedehydrogenase (G6PDHy), pyruvate formate lyase (PFLi), NAD(P)transhydrogenase (THD2), ATP synthetase (ATPS4r), glutamatedehydrogenase (GLUDy), aspartase (ASPT), acetate kinase (ACKr),phosphotransacetylase (PTAr) and NADH dehydrogenase (NADH6). Addition ofany of these knockouts to the strain designs in Tables 6 and 8 or any ofthe non-naturally occurring microbial organisms disclosed herein willfurther improve the yield of MAA or 3-hydroxyisobutryrate.

Example XXV Central Metabolic Enzymes Providing Increased TheoreticalYields of MAA and/or 3-Hydroxyisobutyrate via a Succinyl-CoA or4-Hydroxybutyryl-CoA Precursor Pathway

This example describes enzymes of central metabolic reactions that canbe modulated to increase the theoretical yields of organisms engineeredwith a MAA and/or 3-hydroxyisobutyrate pathway utilizing succinyl-CoA or4-hydroxybutyryl-CoA as a precursor.

In this example, we demonstrate the importance of several centralmetabolic reactions that allow high yields of MAA in an engineeredmicrobe via a succinyl-CoA to MAA pathway or a 4-hydroxybutyryl-CoA toMAA pathway. The analysis described in this example equally applies if3-hydroxyisobutyric acid is produced by the engineered microbe alongwith or instead of MAA. Specifically, a series of linear programming(LP) problems were solved that maximized the MAA, or 3-hydroxyisobutyricacid, yield from glucose for an E. coli metabolic network supplementedwith either or both of the MAA production pathways, assuming that everyreaction in central metabolism was individually deleted. As discussedabove, the maximum MAA yield from glucose via either pathway is 1.33mol/mol. Central metabolism includes all reactions in glycolysis, thepentose phosphate pathway, the tricarboxylic acid cycle, the glyoxylateshunt, and various anapleurotic reactions. Unless otherwise noted, itwas assumed that PEP carboxykinase could operate only in thegluconeogenic, ATP-consuming direction towards phosphoenolpyruvate.Although E. coli was chosen as an exemplary microorganism, the analysispresented herein is applicable to virtually any prokaryotic oreukaryotic organism. Additionally, the conclusions described herein arevalid independent of the exemplary carbohydrate feedstock, arbitrarilychosen in this example to be glucose.

Reactions whose deletion negatively affects the maximum MAA yield in thepresence of an external electron acceptor (for example, oxygen, nitrate)are shown in Table 14 for three network assumptions: 1) undeletedwild-type network (that is, all reactions are present); 2) the wild-typenetwork minus malate dehydrogenase (that is, a reaction targeted forattenuation in several OptKnock designs); and 3) the network minus bothmalate dehydrogenase and pyruvate formate lyase (that is, two reactionstargeted for attenuation in several OptKnock designs). Similar resultsassuming that no external electron acceptor is present are provided inTable 15. This analysis led to three important observations, asdiscussed below in more detail.

TABLE 14 The maximum theoretical MAA molar yields on glucose areprovided assuming that various central metabolic reactions are eachindividually inactivated. The analysis assumes that an external electronacceptor such as oxygen is present and that PEP carboxykinase is notused to produce oxaloacetate. Three cases are explored: 1) WT—wild- typenetwork including all E. coli central metabolic reactions; 2)ΔMDH—wild-type network minus malate dehydrogenase activity; 3) ΔMDH,ΔPFL—wild-type network minus malate dehydrogenase and pyruvate formatelyase activities. WT ΔMDH ΔMDH, ΔPFL % of % of % of MAA Max MAA Max MAAMax Abbreviation Reaction Name Yield Yield Yield Yield Yield Yield ACONTAconitase 1.067 80.0% 0.954 71.6% 0.954 71.6% CS Citrate Synthase 1.06780.0% 0.954 71.6% 0.954 71.6% ENO Enolase 1.132 84.9% 1.097 82.3% 1.09782.3% FUM Fumarase 1.297 97.3% 1.297 97.3% 1.297 97.3% GAPDGlyceraldehyde-3-phosphate 1.132 84.9% 1.097 82.3% 1.097 82.3%Dehydrogenase ICL Isocitrate Lyase 1.333  100% 1.284 96.3% 1.284 96.3%MALS Malate synthase 1.333  100% 1.297 97.3% 1.297 97.3% PDH Pyruvatedehydrogenase 1.333  100% 1.306 97.9% 1.231 92.3% PGIPhosphoglucoisomerase 1.330 99.8% 1.296 97.2% 1.296 97.2% PGKPhosphoglycerate Kinase 1.132 84.9% 1.097 82.3% 1.097 82.3% PGMPhosphoglycerate Mutase 1.132 84.9% 1.097 82.3% 1.097 82.3% PPC PEPcarboxylase 1.200 90.0% 1.163 87.2% 1.163 87.2% TPI Triose PhosphateIsomerase 1.288 96.6% 1.286 96.5% 1.286 96.5%

TABLE 15 The maximum theoretical MAA molar yields on glucose areprovided assuming that various central metabolic reactions are eachindividually inactivated. The analysis assumes that an external electronacceptor such as oxygen is not present and that PEP carboxykinase is notused to produce oxaloacetate. Three cases are explored: 1) WT—wild- typenetwork including all E. coli central metabolic reactions; 2)ΔMDH—wild-type network minus malate dehydrogenase activity, 3) ΔMDH,ΔPFL—wild-type network minus malate dehydrogenase and pyruvate formatelyase activities. WT ΔMDH ΔMDH, ΔPFL % of % of % of MAA Max MAA Max MAAMax Abbreviation Reaction Name Yield Yield Yield Yield Yield Yield ACONTAconitase 1.067 80.0% 0.845 63.4% 0.845 63.4% CS Citrate Synthase 1.06780.0% 0.845 63.4% 0.845 63.4% ENO Enolase 0.000  0.0% 0.000 0.0% 0.0000.0% FUM Fumarase 1.091 81.8% 1.091 81.8% 1.053 78.9% GAPDGlyceraldehyde-3-phosphate 0.000  0.0% 0.000 0.0% 0.000 0.0%dehydrogenase ICL Isocitrate Lyase 1.333  100% 1.033 77.5% 0.990 74.3%MALS Malate synthase 1.333  100% 1.091 81.8% 1.053 78.9% PDH Pyruvatedehydrogenase 1.333  100% 1.277 95.7% 0.770 57.8% PGIPhosphoglucoisomerase 1.317 98.8% 1.014 76.1% 0.909 68.2% PGKPhosphoglycerate Kinase 0.000  0.0% 0.000 0.0% 0.000 0.0% PGMPhosphoglycerate Mutase 0.000  0.0% 0.000 0.0% 0.000 0.0% PPC PEPcarboxylase 0.839 62.9% 0.000 0.0% 0.000 0.0% TPI Triose PhosphateIsomerase 1.108 83.1% 0.988 74.1% 0.909 68.2%

Observation 1. Sufficient flux through citrate synthase and aconitase isrequired to achieve the greater than 80% of the theoretical yield of MAAin all cases. Though highly active under aerobic conditions, theoxidative branch of the tricarboxylic acid cycle is not highly active inthe absence of an external electron acceptor such as oxygen or nitrate.In E. coli, for example, citrate synthase is inhibited by NADH, whoseconcentration is high in the absence of an external electron acceptor.Furthermore, under oxygen-limited conditions, the expression of thetricarboxylic acid cycle enzymes is repressed by product of the arcAgene (Alexeeva, et al., J. Bacteriol. 185(1):204-209 (2003)). Anexemplary method for increasing citrate synthase and aconitase activityin E. coli under oxygen-limited conditions involves deleting theregulator arcA and/or replacing the native citrate synthase with anNADH-insensitive enzyme (Stokell et al., J. Biol. Chem. 278:35435-35443(2003); Jin and Sonenshein, J. Bacteriol. 178(12):3658-3660 (1996).

Observation 2. The glyoxylate shunt enzymes, isocitrate lysase, andmalate synthase, are required to achieve the maximum theoretical yieldof MAA when malate dehydrogenase activity is attenuated. The requirementfor the glyoxylate shunt is exacerbated under oxygen-limited conditionsas the maximum yield of MAA drops approximately 20% without isocitratelysase or malate synthase activities. An exemplary method for increasingglyoxylate shunt activity in E. coli involves deleting thetranscriptional repressor, iclR, as described in Sanchez, et al. (Metab.Eng. 7 (3) 229-239 (2005).

Observation 3. In a malate dehydrogenase and pyruvate formate lyasedeficient background, pyruvate dehydrogenase is required to reach 93% ofthe maximum theoretical MAA yield in the presence of an externalelectron acceptor or 58% of the maximum theoretical yield in the absenceof an external electron acceptor. Pyruvate dehydrogenase is inhibited byhigh NADH/NAD, ATP/ADP, and acetyl-CoA/CoA ratios. Thus the enzymenaturally exhibits very low activity under oxygen-limited or anaerobicconditions in organisms such as E. coli due in large part to the NADHsensitivity of the subunit E3, encoded by lpdA. Exemplary methods forobtaining pyruvate dehydrogenase activity in E. coli underoxygen-limited conditions include replacing the native promoter with ananaerobically-induced promoter (Zhou et al., Biotechnol. Lett.30(2):335-342 (2008)), introducing a point mutation into lpdA to relievethe NADH sensitivity (Kim et al., J. Bacteriol. 190 (11) 3851-3858(2008), or inactivating the repressor, pdhR (Quail and Guest, Mol.Microbiol. 15 (3) 519-529 (1995)). Net pyruvate dehydrogenase-likeactivity can alternatively be obtained from pyruvate ferredoxinoxidoreductase. To do so, a pyruvate ferredoxin oxidoreductase (PFOR)enzyme is used to convert pyruvate to acetyl-CoA with the concaminantreduction of a ferredoxin protein. The reduced ferredoxin then transfersits electrons to NAD+ or NADP+ by way of NAD(P)H/ferredoxinoxidoreductase. Heterologous and native PFOR genes have recently beendemonstrated to improve hydrogen production in E. coli (Akhtar andJones, Metab. Eng. 11:139-147 (2009); Do et al., Appl. Biochem.Biotechnol. 153:21-33 (2009)).

Lastly, the analysis was repeated assuming that PEP carboxykinase canoperate in the ATP-forming, CO₂-fixing direction towards oxaloacetate.In organisms such as E. coli, the metabolic flux fromphosphoenolpyruvate to oxaloacetate is carried by PEP carboxylase, anenzyme that does not generate an ATP equivalent. However, CO₂-fixing PEPcarboxykinase activity can be enhanced in E. coli by overexpressing thenative PEP carboxykinase under the appropriate conditions (Deok et al.,J. Microbiol. Biotechnol. 16 (9) 1448-1452 (2006)) or by expressingforeign genes encoding PEP carboxykinase enzymes with more favorablekinetic properties. The observed PEP carboxykinase activity might bemore prevalent in a host organism with attenuated PEP carboxylaseactivity (Kim et al, Appl. Env. Microbiol. 70 (2) 1238-1241 (2004)).Assuming that PEP carboxykinase can carry a significant net flux towardsoxaloacetate eliminates the absolute requirement for isocitrate lyaseand malate synthase activity to achieve the maximum yield of MAA in allcases. Furthermore, the requirement for pyruvate dehydrogenase is alsoeliminated in the wild-type and malate dehydrogenase negativebackgrounds. Nevertheless, engineering PEP carboxykinase activity intothe host organism chosen for MAA production will be useful due to itsrole in improving the maximum ATP yield of the MAA pathways from 0.47mol/mol to 1.71 mol/mol.

This example describes additional modifications that can be introducedinto a 3-hydroxyisobutyrate or MAA producing microbial organism toincrease product yield.

TABLE 10 Growth-coupled production designs for the succinyl-CoA:MAApathway (FIG. 2). Predicted Design MAA ID Metabolic TransformationsTargeted for Removal Yield 1 FUM 1.31393 2 HEX1 0.81503 3 MDH 0.7159 4PFK and/or FBA and/or TPI 0.32284 5 MDH, THD2 and/or GLUDy 6.09185 6FUM, PFLi 5.98191 7 HEX1, PFLi 5.24339 8 MDH, PFLi 5.21195 9 PFK and/orFBA and/or TPI, PFLi 4.87678 10 ADHEr, PPCK 4.25091 11 ADHEr, FRD and/orSUCD4 4.17475 12 HEX1, THD2 and/or GLUDy 3.09819 13 FUM, HEX1 1.81756 14MDH, PFK and/or FBA and/or TPI 1.36009 15 FRD and/or SUCD4, PFLi 1.0780816 PFLi, PPCK 0.94993 17 PPCK, PYK 0.57249 18 ADHEr, PFLi, PPCK 6.9352819 ADHEr, FRD and/or SUCD4, PFLi 6.8792 20 HEX1, PFLi, THD2 and/or GLUDy6.71657 21 MDH, PFK and/or FBA and/or TPI, PFLi 6.3322 22 MDH, PFLi,THD2 and/or GLUDy 6.21103 23 FUM, ME2, THD2 and/or GLUDy 6.09185 24PFLi, PPCK, PYK 5.16721 25 ADHEr, PPCK, THD2 and/or GLUDy 4.91251 26ADHEr, PFK and/or FBA and/or TPI, PPCK 4.61324 27 ADHEr, HEX1, PFKand/or FBA and/or TPI 4.5815 28 ADHEr, FRD and/or SUCD4, PFK and/or FBAand/or TPI 4.57316 29 ADHEr, MDH, THD2 and/or GLUDy 4.35906 30 ADHEr,FRD and/or SUCD4, MDH 4.3526 31 ADHEr, GLCpts, PPCK 4.33751 32 ADHEr,HEX1, THD2 and/or GLUDy 4.32305 33 ADHEr, MDH, PPCK 4.3218 34 ADHEr,FUM, PPCK 4.3218 35 ADHEr, FRD and/or SUCD4, ME2 4.27691 36 ADHEr, FUM,THD2 and/or GLUDy 4.26872 37 ADHEr, FRD and/or SUCD4, THD2 and/or GLUDy4.26122 38 ADHEr, FRD and/or SUCD4, GLCpts 4.23155 39 ADHEr, FUM, HEX14.07963 40 GLUDy, HEX1, THD2 and/or GLUDy 3.74821 41 ME2, PGL and/orG6PDHy, THD2 and/or GLUDy 3.74546 42 HEX1, ME2, THD2 and/or GLUDy3.17934 43 MDH, PYK, THD2 and/or GLUDy 3.01298 44 MDH, PPCK, PYK 2.8896645 FUM, PPCK, PYK 2.88966 46 PPCK, PYK, THD2 and/or GLUDy 2.28488 47PFLi, PPCK, THD2 and/or GLUDy 1.92036 48 ACKr and/or PTAr, FRD and/orSUCD4, PFLi 1.19121 49 ADHEr, MDH, PGL and/or G6PDHy, THD2 and/or GLUDy10.50357 50 ADHEr, MDH, PFLi, THD2 and/or GLUDy 8.26017 51 ADHEr, PFKand/or FBA and/or TPI, PFLi, PPCK 7.5749 52 ADHEr, FRD and/or SUCD4, PFKand/or FBA and/or TPI, PFLi 7.49524 53 ADHEr, HEX1, PFK and/or FBAand/or TPI, PFLi 7.47549 54 ADHEr, PFLi, PPCK, THD2 and/or GLUDy 7.3244855 HEX1, ME2, PGL and/or G6PDHy, THD2 and/or GLUDy 7.30328 56 ADHEr,GLCpts, PFLi, PPCK 7.07538 57 ADHEr, HEX1, PFLi, THD2 and/or GLUDy7.04634 58 ADHEr, FRD and/or SUCD4, ME2, PFLi 7.04349 59 GLUDy, HEX1,PFLi, THD2 and/or GLUDy 7.02387 60 ADHEr, FRD and/or SUCD4, PFLi, THD2and/or GLUDy 6.99958 61 ADHEr, ASPT, LDH_D, MDH 6.91371 62 PFLi, PPCK,PYK, THD2 and/or GLUDy 6.78153 63 ADHEr, FUM, HEX1, PFLi 6.65795 64 FUM,ME2, PFK and/or FBA and/or TPI, PFLi 6.3322 65 ADHEr, FRD and/or SUCD4,ME2, THD2 and/or GLUDy 6.21914 66 FUM, ME2, PFLi, THD2 and/or GLUDy6.21103 67 ADHEr, GLUDy, MDH, THD2 and/or GLUDy 6.18117 68 ADHEr, MDH,PPCK, THD2 and/or GLUDy 6.17362 69 ADHEr, FUM, PPCK, THD2 and/or GLUDy6.17362 70 ME2, PFLi, PGL and/or G6PDHy, THD2 and/or GLUDy 6.01239 71ADHEr, ASPT, MDH, PYK 5.92643 72 ADHEr, FRD and/or SUCD4, PFK and/or FBAand/or TPI, PPCK 4.76879 73 ADHEr, HEX1, PFK and/or FBA and/or TPI, PPCK4.76303 74 ADHEr, FRD and/or SUCD4, HEX1, PFK and/or FBA and/or TPI4.73051 75 ADHEr, FUM, PFK and/or FBA and/or TPI, THD2 and/or GLUDy4.63551 76 ADHEr, MDH, PFK and/or FBA and/or TPI, THD2 and/or GLUDy4.63551 77 ADHEr, FRD and/or SUCD4, PPCK, PYK 4.53921 78 ADHEr, FRDand/or SUCD4, LDH_D, PPCK 4.4635 79 ADHEr, FRD and/or SUCD4, GLCpts, MDH4.44642 80 ADHEr, GLCpts, MDH, THD2 and/or GLUDy 4.44284 81 ADHEr, MDH,PYK, THD2 and/or GLUDy 4.42534 82 ADHEr, FUM, GLCpts, PPCK 4.41046 83ADHEr, GLCpts, MDH, PPCK 4.41046 84 ADHEr, GLCpts, PPCK, THD2 and/orGLUDy 4.40279 85 ASPT, MDH, PGL and/or G6PDHy, PYK 4.3931 86 ADHEr, MDH,PPCK, PYK 4.39083 87 ADHEr, FUM, PPCK, PYK 4.39083 88 ADHEr, FRD and/orSUCD4, GLCpts, ME2 4.36844 89 ADHEr, FUM, ME2, THD2 and/or GLUDy 4.3590690 ADHEr, FRD and/or SUCD4, FUM, ME2 4.3526 91 ADHEr, FUM, GLCpts, THD2and/or GLUDy 4.32647 92 ADHEr, FRD and/or SUCD4, GLCpts, THD2 and/orGLUDy 4.31559 93 FRD and/or SUCD4, FUM, PFK and/or FBA and/or TPI, THD54.08513 94 FRD and/or SUCD4, MDH, PFK and/or FBA and/or TPI, THD54.08513 95 ACKr and/or PTAr, ME2, PGL and/or G6PDHy, THD2 and/or GLUDy3.89111 96 PGL and/or G6PDHy, PPCK, PYK, THD2 and/or GLUDy 3.46752 97FUM, HEX1, PFK and/or FBA and/or TPI, THD5 3.35722 98 HEX1, MDH, PFKand/or FBA and/or TPI, THD5 3.35722 99 FRD and/or SUCD4, ME2, PFLi, THD2and/or GLUDy 2.78398 100 FRD and/or SUCD4, ME1x, ME2, PYK 2.6437 101ACKr and/or PTAr, PFLi, PPCK, THD2 and/or GLUDy 2.01602 102 FRD and/orSUCD4, FUM, MDH, PYK 1.89207 103 ACKr and/or PTAr, ME2, PGL and/orG6PDHy, SUCOAS 1.83792 104 FUM, GLYCL, ME2, PFK and/or FBA and/or TPI1.36495 105 ACKr and/or PTAr, FRD and/or SUCD4, GLU5K, PFLi 1.24122 106ACKr and/or PTAr, FRD and/or SUCD4, G5SD, PFLi 1.24122 107 ACKr and/orPTAr, GLU5K, PFLi, PPCK 1.09336 108 ACKr and/or PTAr, G5SD, PFLi, PPCK1.09336 109 ACKr and/or PTAr, AKGD, PFLi, PPCK 1.04907 110 ACKr and/orPTAr, ME2, PFLi, PPCK 1.04907 111 ACKr and/or PTAr, LDH_D, PFLi, PPCK1.04907 112 ACKr and/or PTAr, PFLi, PGL and/or G6PDHy, PPCK 1.04907 113ACKr and/or PTAr, ASPT, PFLi, PPCK 1.04907 114 ACKr and/or PTAr, PFLi,PPCK 1.04907 115 ACKr and/or PTAr, ACS, PFLi, PPCK 1.04907 116 ACKrand/or PTAr, ADHEr, ASPT, MDH 0.91363 117 FRD and/or SUCD4, PFK and/orFBA and/or TPI, THD2 and/or 0.79247 GLUDy, THD5 118 ADHEr, AKGD, ASPT,MDH 0.7853 119 ADHEr, ASPT, MDH, P5CD 0.7853 120 ADHEr, ASPT, MDH, PGLand/or G6PDHy 0.7853 121 ADHEr, ASPT, MDH, PDH 0.7853 122 ADHEr, ASPT,MDH, VALTA 0.7853 123 ADHEr, ASPT, MDH, ME2 0.7853 124 ADHEr, ASPT, MDH,PPS 0.7853 125 ADHEr, ASPT, MDH, NACODA 0.7853 126 ADHEr, ASPT, MDH0.7853 127 ADHEr, ASPT, LDH_D, MDH, PFLi 11.64516 128 ACKr and/or PTAr,ADHEr, FRD and/or SUCD4, LDH_D, ME2 10.90737 129 ADHEr, FUM, ME2, PGLand/or G6PDHy, THD2 and/or GLUDy 10.88038 130 ADHEr, ICL, MDH, PGLand/or G6PDHy, THD2 and/or GLUDy 10.88038 131 ADHEr, MALS, MDH, PGLand/or G6PDHy, THD2 and/or GLUDy 10.88038 132 ASPT, MDH, PGL and/orG6PDHy, PYK, SERD_L 10.86679 133 ADHEr, GLCpts, MDH, PGL and/or G6PDHy,THD2 and/or GLUDy 10.79871 134 ADHEr, ASPT, MDH, PGL and/or G6PDHy, PYK10.7622 135 ADHEr, FRD and/or SUCD4, ME2, PGL and/or G6PDHy, THD2 and/or10.51703 GLUDy 136 ASPT, MDH, PGL and/or G6PDHy, PYK, THD2 and/or GLUDy10.0408 137 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, MDH9.09361 138 MDH, ME2, PGL and/or G6PDHy, PYK, THD2 and/or GLUDy 8.70446139 ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH 8.58714 140 ACKr and/orPTAr, ADHEr, LDH_D, MDH, THD2 and/or GLUDy 8.35695 141 FUM, MDH, PGLand/or G6PDHy, PYK, THD2 and/or GLUDy 8.28079 142 ADHEr, FUM, ME2, PFLi,THD2 and/or GLUDy 8.26017 143 HEX1, ME2, PFLi, PGL and/or G6PDHy, THD2and/or GLUDy 7.86496 144 ADHEr, ASPT, LDH_D, MDH, THD2 and/or GLUDy7.77845 145 ADHEr, FUM, PFK and/or FBA and/or TPI, PFLi, THD2 and/or7.59996 GLUDy 146 ADHEr, MDH, PFK and/or FBA and/or TPI, PFLi, THD2and/or 7.59996 GLUDy 147 ADHEr, FRD and/or SUCD4, PFK and/or FBA and/orTPI, PFLi, THD2 7.55146 and/or GLUDy 148 ADHEr, HEX1, PFK and/or FBAand/or TPI, PFLi, THD2 and/or 7.5299 GLUDy 149 ADHEr, FRD and/or SUCD4,LDH_D, PFLi, PPCK 7.51427 150 ADHEr, GLCpts, PFLi, PPCK, THD2 and/orGLUDy 7.41336 151 ADHEr, GLUDy, PFLi, PPCK, THD2 and/or GLUDy 7.39109152 ADHEr, ASPT, MDH, PYK, THD2 and/or GLUDy 7.30613 153 ADHEr, ASPT,FRD and/or SUCD4, LDH_D, MDH 7.2706 154 ADHEr, FRD and/or SUCD4, LDH_D,MDH, PFLi 7.25565 155 ADHEr, MDH, PFLi, PYK, THD2 and/or GLUDy 7.21719156 ADHEr, ASPT, LDH_D, MDH, PPCK 7.20783 157 ADHEr, FRD and/or SUCD4,GLCpts, ME2, PFLi 7.19295 158 ADHEr, LDH_D, MDH, PFLi, THD2 and/or GLUDy7.17902 159 ADHEr, FUM, PFLi, PPCK, PYK 7.16927 160 ADHEr, MDH, PFLi,PPCK, PYK 7.16927 161 ADHEr, FRD and/or SUCD4, ME2, PFLi, THD2 and/orGLUDy 7.14501 162 ADHEr, GLUDy, HEX1, PFLi, THD2 and/or GLUDy 7.13398163 ADHEr, FUM, LDH_D, PFLi, PPCK 7.12989 164 ADHEr, LDH_D, MDH, PFLi,PPCK 7.12989 165 ADHEr, ASPT, GLCpts, LDH_D, MDH 7.08892 166 ADHEr, FRDand/or SUCD4, FUM, LDH_D, PFLi 7.06106 167 ADHEr, FUM, GLCpts, PFLi,THD2 and/or GLUDy 7.05969 168 ADHEr, FUM, LDH_D, PFLi, THD2 and/or GLUDy7.03327 169 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PFLi 7.00666 170ADHEr, NADH6 5.44845 171 ADHEr, ATPS4r 2.36532 172 ADHEr, PGI 1.80553173 ADHEr, FUM 1.31393 174 ADHEr, HEX1 0.81503 175 ADHEr, MDH 0.7159 176ADHEr, PFK and/or FBA and/or TPI 0.32284 177 ADHEr, HEX1, PGI 8.63121178 ADHEr, NADH6, PFLi 6.77656 179 ADHEr, NADH6, PGI 6.11877 180 ADHEr,NADH6, PFK and/or FBA and/or TPI 6.01968 181 ADHEr, FUM, PFLi 5.98191182 ADHEr, NADH6, PPCK 5.82769 183 ADHEr, MDH, NADH6 5.64458 184 ADHEr,NADH6, THD2 and/or GLUDy 5.57367 185 ADHEr, FUM, NADH6 5.51162 186ADHEr, HEX1, PFLi 5.24339 187 ADHEr, MDH, PFLi 5.21195 188 ADHEr, PFKand/or FBA and/or TPI, PFLi 4.87678 189 ADHEr, ATPS4r, PPCK 4.69887 190ADHEr, PGI, PPCK 4.67315 191 ADHEr, FRD and/or SUCD4, PGI 4.63924 192ADHEr, ATPS4r, MDH 3.93602 193 ADHEr, ATPS4r, THD2 and/or GLUDy 3.20207194 ADHEr, ATPS4r, FUM 2.70933 195 ADHEr, PFLi, PGI 2.48299 196 ADHEr,MDH, PFK and/or FBA and/or TPI 1.36009 197 ADHEr, HEX1, PFLi, PGI9.89317 198 ADHEr, HEX1, PGI, THD2 and/or GLUDy 8.685 199 ADHEr, MDH,NADH6, THD2 and/or GLUDy 8.42455 200 ADHEr, PFLi, PGI, PPCK 7.60434 201ADHEr, NADH6, PFLi, PGI 7.53021 202 ADHEr, FRD and/or SUCD4, PFLi, PGI7.53021 203 ADHEr, NADH6, PFK and/or FBA and/or TPI, PFLi 7.49524 204ADHEr, ATPS4r, MDH, NADH6 7.09625 205 ADHEr, MDH, NADH6, PFLi 7.03739206 ACKr and/or PTAr, ADHEr, NADH6, PGI 7.02293 207 ADHEr, NADH6, PFLi,THD2 and/or GLUDy 6.90622 208 ADHEr, GLCpts, NADH6, PFLi 6.89924 209ADHEr, NADH12, NADH6, PFLi 6.8792 210 ADHEr, FUM, NADH6, PFLi 6.87559211 ADHEr, ME2, NADH6, PFLi 6.83907 212 ACKr and/or PTAr, ADHEr, FRDand/or SUCD4, PGI 6.83058 213 ADHEr, ATPS4r, NADH6, PGI 6.62593 214ADHEr, NADH6, PPCK, THD2 and/or GLUDy 6.57106 215 ADHEr, ATPS4r, NADH6,PFK and/or FBA and/or TPI 6.48882 216 ADHEr, NADH6, PGI, PPCK 6.4075 217ADHEr, NADH6, PFK and/or FBA and/or TPI, PPCK 6.35839 218 ADHEr, MDH,PFK and/or FBA and/or TPI, PFLi 6.3322 219 ADHEr, ATPS4r, FUM, NADH66.33033 220 ADHEr, ME2, NADH6, THD2 and/or GLUDy 6.30041 221 ADHEr,HEX1, NADH6, PFK and/or FBA and/or TPI 6.28787 222 ADHEr, NADH6, PGI,THD2 and/or GLUDy 6.17721 223 ADHEr, NADH6, PFK and/or FBA and/or TPI,THD2 and/or GLUDy 6.08946 224 ADHEr, ATPS4r, NADH6, PPCK 5.95899 225ADHEr, GLCpts, NADH6, PPCK 5.94641 226 ADHEr, NADH6, PPCK, PYK 5.88622227 ADHEr, GLCpts, MDH, NADH6 5.76626 228 ADHEr, ATPS4r, GLCpts, PPCK5.74112 229 ADHEr, FUM, ME2, NADH6 5.64458 230 ADHEr, FUM, HEX1, NADH65.59255 231 ADHEr, ATPS4r, HEX1, NADH6 5.58729 232 ADHEr, HEX1, NADH6,THD2 and/or GLUDy 5.50758 233 ADHEr, ATPS4r, MDH, THD2 and/or GLUDy5.42607 234 ADHEr, ATPS4r, FUM, PPCK 5.41736 235 ADHEr, ATPS4r, MDH,PPCK 5.41736 236 ADHEr, ATPS4r, MDH, PGL and/or G6PDHy 5.3991 237 ADHEr,ATPS4r, PGI, PPCK 5.39847 238 ADHEr, ATPS4r, PFK and/or FBA and/or TPI,PPCK 5.2252 239 ADHEr, ATPS4r, FUM, HEX1 5.09544 240 ADHEr, ATPS4r, PGLand/or G6PDHy, PPCK 5.02209 241 ADHEr, PFK and/or FBA and/or TPI, PFLi,PGI 5.01176 242 ADHEr, ATPS4r, PFLi, PGI 5.00885 243 ADHEr, ATPS4r, ME2,THD2 and/or GLUDy 4.89177 244 ADHEr, ATPS4r, FUM, THD2 and/or GLUDy4.82795 245 ADHEr, FRD and/or SUCD4, PGI, PPCK 4.80562 246 ADHEr, FUM,PGI, THD2 and/or GLUDy 4.69172 247 ADHEr, MDH, PGI, THD2 and/or GLUDy4.69172 248 ADHEr, ATPS4r, FUM, ME2 3.93602 249 ADHEr, ME2, PGL and/orG6PDHy, THD2 and/or GLUDy 3.74546 250 ACKr and/or PTAr, ADHEr, ATPS4r,SUCOAS 3.23462 251 ADHEr, ASNS2, ATPS4r, GLU5K 2.42406 252 ADHEr, ASNS2,ATPS4r, G5SD 2.42406 253 ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH611.12044 254 ADHEr, ATPS4r, MDH, PGL and/or G6PDHy, THD2 and/or GLUDy10.65458 255 ADHEr, HEX1, PFLi, PGI, THD2 and/or GLUDy 9.97214 256ADHEr, ATPS4r, GLCpts, MDH, PGL and/or G6PDHy 9.83354 257 ADHEr, ATPS4r,GLCpts, NADH6, PFLi 9.61783 258 ADHEr, ME2, NADH6, PGL and/or G6PDHy,THD2 and/or GLUDy 8.74922 259 ADHEr, GLCpts, MDH, NADH6, THD2 and/orGLUDy 8.51047 260 ADHEr, FUM, ME2, NADH6, THD2 and/or GLUDy 8.42455 261ADHEr, ATPS4r, MDH, NADH6, PGL and/or G6PDHy 8.35879 262 ADHEr, ATPS4r,MDH, PDH, PGL and/or G6PDHy 8.19203 263 ADHEr, ATPS4r, GLCpts, MDH,NADH6 8.11809 264 ADHEr, ASPT, ATPS4r, LDH_D, MDH 8.05129 265 ADHEr,ASPT, ATPS4r, MDH, PYK 7.89307 266 ADHEr, ASPT, ATPS4r, GLCpts, MDH7.76592 267 ADHEr, ATPS4r, LDH_D, NADH6, PFLi 7.66468 268 ADHEr, FUM,PFLi, PGI, THD2 and/or GLUDy 7.62739 269 ADHEr, MDH, PFLi, PGI, THD2and/or GLUDy 7.62739 270 ADHEr, NADH6, PFLi, PGI, THD2 and/or GLUDy7.58195 271 ADHEr, FRD and/or SUCD4, PFLi, PGI, THD2 and/or GLUDy7.58195 272 ADHEr, NADH6, PFK and/or FBA and/or TPI, PFLi, THD2 and/or7.55146 GLUDy 273 ADHEr, ATPS4r, ME2, PGL and/or G6PDHy, THD2 and/orGLUDy 7.45944 274 ACKr and/or PTAr, ADHEr, ATPS4r, NADH6, PGI 7.37787275 ADHEr, NADH6, PFLi, PPCK, PYK 7.33669 276 ADHEr, HEX1, ME2, PGLand/or G6PDHy, THD2 and/or GLUDy 7.30328 277 ADHEr, LDH_D, NADH6, PFLi,PPCK 7.29288 278 ADHEr, ME2, NADH6, PFLi, THD2 and/or GLUDy 7.26202 279ADHEr, GLCpts, MDH, NADH6, PFLi 7.1878 280 ADHEr, ATPS4r, ME2, NADH6,PFLi 7.18544 281 ADHEr, ASPT, LDH_D, MDH, NADH6 7.1375 282 ADHEr,ATPS4r, FUM, ME2, NADH6 7.09625 283 ADHEr, ME2, NADH12, NADH6, PFLi7.04349 284 ADHEr, FUM, ME2, NADH6, PFLi 7.03739 285 ADHEr, GLCpts,NADH6, PFLi, THD2 and/or GLUDy 7.02149 286 ADHEr, ATPS4r, GLCpts, NADH6,PPCK 7.00602 287 ADHEr, ASPT, MDH, PFLi, PGL and/or G6PDHy, PYK 12.62367288 ADHEr, ATPS4r, GLCpts, MDH, NADH6, PGL and/or G6PDHy 12.58702 289ACKr and/or PTAr, ADHEr, LDH_D, MDH, PFLi, THD2 and/or 12.17542 GLUDy290 ADHEr, ASPT, GLCpts, LDH_D, MDH, PFLi 12.14168 291 ADHEr, ASPT,LDH_D, MDH, PFLi, THD2 and/or GLUDy 11.92294 292 ADHEr, ASPT, MDH,NADH6, PGL and/or G6PDHy, PYK 11.86695 293 ACKr and/or PTAr, ADHEr, FRDand/or SUCD4, LDH_D, MDH, THD2 11.81945 and/or GLUDy 294 ADHEr, ASPT,LDH_D, MDH, PFLi, PYK 11.70177 295 ADHEr, ASPT, FRD and/or SUCD4, MDH,PGL and/or G6PDHy, PYK 11.69597 296 ACKr and/or PTAr, ADHEr, FRD and/or11.67923 SUCD4, GLCpts, LDH_D, MDH 297 ACKr and/or PTAr, ADHEr, LDH_D,MDH, NADH6, THD2 and/or 11.60977 GLUDy 298 ACKr and/or PTAr, ADHEr,GLCpts, LDH_D, MDH, NADH6 11.4429 299 ADHEr, ASPT, MDH, PGL and/orG6PDHy, PYK, THD2 and/or 11.34596 GLUDy 300 ADHEr, ATPS4r, GLCpts, MDH,PGL and/or G6PDHy, THD2 and/or 11.31505 GLUDy 301 ADHEr, ATPS4r, MDH,NADH6, PGL and/or G6PDHy, THD2 and/or 11.27165 GLUDy 302 ACKr and/orPTAr, ADHEr, ATPS4r, GLCpts, MDH, NADH6 11.21212 303 ADHEr, LDH_D, NADH65.44845 304 ADHEr, LDH_D, PPCK 4.25091 305 ADHEr, FRD and/or SUCD4,LDH_D 4.17475 306 ADHEr, ATPS4r, LDH_D 2.36532 307 ADHEr, LDH_D, PGI1.80553 308 ADHEr, FUM, LDH_D 1.31393 309 ADHEr, HEX1, LDH_D 0.81503 310ADHEr, LDH_D, MDH 0.7159 311 ADHEr, LDH_D, PFK and/or FBA and/or TPI0.32284 312 ADHEr, HEX1, LDH_D, PGI 8.63121 313 ADHEr, LDH_D, PFLi, PPCK6.93528 314 ADHEr, FRD and/or SUCD4, LDH_D, PFLi 6.8792 315 ADHEr,LDH_D, NADH6, PFLi 6.77656 316 ADHEr, LDH_D, NADH6, PGI 6.11877 317ADHEr, LDH_D, MDH, THD2 and/or GLUDy 6.11538 318 ADHEr, LDH_D, NADH6,PFK and/or FBA and/or TPI 6.01968 319 ADHEr, FUM, LDH_D, PFLi 5.98191320 ADHEr, LDH_D, NADH6, PPCK 5.82769 321 ADHEr, LDH_D, MDH, NADH65.64458 322 ADHEr, LDH_D, NADH6, THD2 and/or GLUDy 5.57367 323 ADHEr,FUM, LDH_D, NADH6 5.51162 324 ADHEr, HEX1, LDH_D, PFLi 5.24339 325ADHEr, LDH_D, MDH, PFLi 5.21195 326 ADHEr, LDH_D, PPCK, THD2 and/orGLUDy 4.91251 327 ADHEr, LDH_D, PFK and/or FBA and/or TPI, PFLi 4.87678328 ADHEr, ATPS4r, LDH_D, PPCK 4.69887 329 ADHEr, LDH_D, PGI, PPCK4.67315 330 ADHEr, FRD and/or SUCD4, LDH_D, PGI 4.63924 331 ADHEr,LDH_D, PFK and/or FBA and/or TPI, PPCK 4.61324 332 ADHEr, HEX1, LDH_D,PFK and/or FBA and/or TPI 4.5815 333 ADHEr, FRD and/or SUCD4, LDH_D, PFKand/or FBA and/or TPI 4.57316 334 ADHEr, FRD and/or SUCD4, LDH_D, MDH4.3526 335 ADHEr, GLCpts, LDH_D, PPCK 4.33751 336 ADHEr, HEX1, LDH_D,THD2 and/or GLUDy 4.32305 337 ADHEr, LDH_D, MDH, PPCK 4.3218 338 ADHEr,FUM, LDH_D, PPCK 4.3218 339 ADHEr, FRD and/or SUCD4, LDH_D, ME2 4.27691340 ADHEr, FUM, LDH_D, THD2 and/or GLUDy 4.26872 341 ADHEr, FRD and/orSUCD4, LDH_D, THD2 and/or GLUDy 4.26122 342 ADHEr, FRD and/or SUCD4,GLCpts, LDH_D 4.23155 343 ADHEr, FUM, HEX1, LDH_D 4.07963 344 ADHEr,ATPS4r, LDH_D, MDH 3.93602 345 ADHEr, ATPS4r, LDH_D, THD2 and/or GLUDy3.20207 346 ADHEr, ATPS4r, FUM, LDH_D 2.70933 347 ADHEr, LDH_D, PFLi,PGI 2.48299 348 ADHEr, LDH_D, MDH, PFK and/or FBA and/or TPI 1.36009 349ADHEr, LDH_D, MDH, PGL and/or G6PDHy, THD2 and/or GLUDy 10.50357 350ADHEr, HEX1, LDH_D, PFLi, PGI 9.89317 351 ADHEr, HEX1, LDH_D, PGI, THD2and/or GLUDy 8.685 352 ADHEr, LDH_D, MDH, NADH6, THD2 and/or GLUDy8.42455 353 ADHEr, LDH_D, PFLi, PGI, PPCK 7.60434 354 ADHEr, LDH_D, PFKand/or FBA and/or TPI, PFLi, PPCK 7.5749 355 ADHEr, LDH_D, NADH6, PFLi,PGI 7.53021 356 ADHEr, FRD and/or SUCD4, LDH_D, PFLi, PGI 7.53021 357ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, PFLi 7.49524 358 ADHEr,FRD and/or SUCD4, LDH_D, PFK and/or FBA and/or 7.49524 TPI, PFLi 359ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI, PFLi 7.47549 360 ADHEr,LDH_D, PFLi, PPCK, THD2 and/or GLUDy 7.32448 361 ADHEr, ATPS4r, LDH_D,MDH, NADH6 7.09625 362 ADHEr, GLCpts, LDH_D, PFLi, PPCK 7.07538 363ADHEr, HEX1, LDH_D, PFLi, THD2 and/or GLUDy 7.04634 364 ADHEr, FRDand/or SUCD4, LDH_D, ME2, PFLi 7.04349 365 ADHEr, LDH_D, MDH, NADH6,PFLi 7.03739 366 ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PGI 7.02293 367ACKr and/or PTAr, ADHEr, FUM, LDH_D, ME2, NADH6 11.12044 368 ADHEr, FRDand/or SUCD4, LDH_D, ME2, PGL and/or 10.92104 G6PDHy, THD2 and/or GLUDy369 ADHEr, FUM, LDH_D, ME2, PGL and/or G6PDHy, THD2 and/or 10.88038GLUDy 370 ADHEr, ICL, LDH_D, MDH, PGL and/or G6PDHy, THD2 and/or10.88038 GLUDy 371 ADHEr, LDH_D, MALS, MDH, PGL and/or G6PDHy, THD2and/or 10.88038 GLUDy 372 ADHEr, GLCpts, LDH_D, MDH, PGL and/or G6PDHy,THD2 and/or 10.79871 GLUDy 373 ADHEr, LDH_D, MDH, NADH6, PGL and/orG6PDHy, THD2 and/or 10.79806 GLUDy 374 ADHEr, ASPT, LDH_D, MDH, PGLand/or G6PDHy, PYK 10.7622 375 ADHEr, ATPS4r, LDH_D, MDH, PGL and/orG6PDHy, THD2 and/or 10.65458 GLUDy 376 ACKr and/or PTAr, ADHEr, LDH_D,MALS, MDH, THD2 and/or 10.65175 GLUDy 377 ACKr and/or PTAr, ADHEr, FUM,LDH_D, ME2, THD2 and/or GLUDy 10.65175 378 ACKr and/or PTAr, ADHEr, ICL,LDH_D, MDH, THD2 and/or GLUDy 10.65175 379 ACKr and/or PTAr, ADHEr, FUM,LDH_D, MDH, THD2 and/or 10.65175 GLUDy 380 ADHEr, FRD and/or SUCD4,LDH_D, MDH, PFLi, THD2 and/or 10.5877 GLUDy 381 ADHEr, ASPT, ATPS4r,GLCpts, LDH_D, MDH 10.28675 382 ADHEr, FRD and/or SUCD4, LDH_D, MDH,PFLi, PGI 10.27254 383 ADHEr, FRD and/or SUCD4, FUM, LDH_D, PFLi, PGI10.24846 384 ADHEr, HEX1, LDH_D, PFLi, PGI, THD2 and/or GLUDy 9.97214385 ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PGL and/or G6PDHy 9.83354 386ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH6 9.76182 387 ADHEr, ATPS4r,GLCpts, LDH_D, NADH6, PFLi 9.61783 388 ADHEr, ATPS4r, LDH_D, MDH, NADH6,PGL and/or G6PDHy 9.57049 389 ACKr and/or PTAr, ADHEr, LDH_D, MDH, PYK,THD2 and/or 9.52381 GLUDy 390 ACKr and/or PTAr, ADHEr, CITL, LDH_D,NADH12, NADH6 9.3809 391 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4,LDH_D, MDH, PFLi 9.27557 392 ADHEr, ATPS4r, LDH_D, MDH, PDH, PGL and/orG6PDHy 9.21865 393 ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6 9.04167 394ADHEr, FRD and/or SUCD4, LDH_D, PFLi, PPCK, THD2 and/or 9.01487 GLUDy395 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi 8.9614 396 ADHEr, ATPS4r,LDH_D, NADH12, NADH6, PFLi 8.93851 397 ADHEr, FRD and/or SUCD4, HEX1,LDH_D, MDH, THD2 and/or 8.89295 GLUDy 398 ADHEr, LDH_D, ME2, NADH6, PGLand/or G6PDHy, THD2 and/or 8.74922 GLUDy 399 ADHEr, GLUDy, LDH_D, MDH,PFLi, THD2 and/or GLUDy 8.69116 400 ACKr and/or PTAr, ADHEr, FUM, HEX1,LDH_D, NADH6 8.68896 401 ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGL and/orG6PDHy 8.68776 402 ADHEr, FUM, LDH_D, PFLi, PPCK, THD2 and/or GLUDy 8.66403 ADHEr, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy 8.66 404 ADHEr, FRDand/or SUCD4, LDH_D, ME2, PFLi, THD2 and/or 8.6194 GLUDy 405 ADHEr,LDH_D, MDH, NADH6, PFLi, THD2 and/or GLUDy 8.58651 406 ADHEr, ATPS4r,LDH_D, MDH, PFLi, THD2 and/or GLUDy 8.54512 407 ADHEr, GLCpts, LDH_D,MDH, NADH6, THD2 and/or GLUDy 8.51047 408 ADHEr, FUM, LDH_D, ME2, NADH6,THD2 and/or GLUDy 8.42455 409 ACKr and/or PTAr, ADHEr, CITL, HEX1,LDH_D, NADH6 8.38082 410 ADHEr, HEX1, LDH_D, MDH, PFLi, THD2 and/orGLUDy 8.33314 411 ADHEr, FUM, HEX1, LDH_D, PFLi, THD2 and/or GLUDy8.33314 412 ADHEr, FUM, LDH_D, ME2, PFLi, THD2 and/or GLUDy 8.26017 413ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, NADH6 8.20955 414 ADHEr,ASPT, ATPS4r, LDH_D, MDH, PPCK 8.16498 415 ADHEr, ATPS4r, GLCpts, LDH_D,MDH, NADH6 8.11809 416 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPCK 7.80331417 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPS 7.78081 418 ADHEr, ASPT,LDH_D, MDH, PGI, THD2 and/or GLUDy 7.70411 419 ADHEr, ASPT, LDH_D, MDH,PFK and/or FBA and/or TPI, THD2 7.69846 and/or GLUDy 420 ADHEr, FRDand/or SUCD4, LDH_D, MDH, PFK and/or FBA and/or 7.69078 TPI, PFLi 421ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, PFLi, PPCK 7.66606 422 ADHEr,FRD and/or SUCD4, FUM, LDH_D, PFK and/or FBA and/or 7.66287 TPI, PFLi423 ADHEr, LDH_D, MDH, PFLi, PGI, THD2 and/or GLUDy 7.62739 424 ADHEr,FUM, LDH_D, PFLi, PGI, THD2 and/or GLUDy 7.62739 425 ACKr and/or PTAr,ADHEr, LDH_D, ME2, NADH12, NADH6 7.61395 426 ADHEr, FUM, LDH_D, PFKand/or FBA and/or TPI, PFLi, THD2 7.59996 and/or GLUDy 427 ADHEr, LDH_D,MDH, PFK and/or FBA and/or TPI, PFLi, THD2 7.59996 and/or GLUDy 428ADHEr, FRD and/or SUCD4, LDH_D, PFLi, PGI, THD2 and/or GLUDy 7.58195 429ADHEr, LDH_D, NADH6, PFLi, PGI, THD2 and/or GLUDy 7.58195 430 ADHEr,ASPT, FRD and/or SUCD4, LDH_D, MDH, PPCK 7.57014 431 ADHEr, FRD and/orSUCD4, LDH_D, PFLi, PPCK, PYK 7.56477 432 ADHEr, LDH_D, NADH6, PFLi,PPCK, THD2 and/or GLUDy 7.55966 433 ADHEr, FRD and/or SUCD4, LDH_D, PFKand/or FBA and/or 7.55146 TPI, PFLi, THD2 and/or GLUDy 434 ADHEr, LDH_D,NADH6, PFK and/or FBA and/or TPI, PFLi, THD2 7.55146 and/or GLUDy 435ADHEr, ATPS4r, LDH_D, PFLi, PPCK, THD2 and/or GLUDy 7.5467 436 ADHEr,HEX1, LDH_D, PFK and/or FBA and/or TPI, PFLi, THD2 7.5299 and/or GLUDy437 ADHEr, LDH_D, NADH12, NADH6, PFLi, PPCK 7.51427 438 ADHEr, FRDand/or SUCD4, GLUDy, LDH_D, MDH, THD2 and/or 7.51308 GLUDy 439 ADHEr,LDH_D, MDH, NADH6, PFLi, PPCK 7.51113 440 ADHEr, FUM, LDH_D, NADH6,PFLi, PPCK 7.51113 441 ADHEr, ATPS4r, LDH_D, ME2, PGL and/or G6PDHy,THD2 and/or 7.45944 GLUDy 442 ADHEr, ASPT, FRD and/or SUCD4, GLCpts,LDH_D, MDH 7.45295 443 ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK 7.44019444 ADHEr, ASPT, LDH_D, MDH, NADH6, PPCK 7.43508 445 ADHEr, GLCpts,LDH_D, PFLi, PPCK, THD2 and/or GLUDy 7.41336 446 ADHEr, FRD and/orSUCD4, GLCpts, LDH_D, MDH, PFLi 7.41073 447 ADHEr, FRD and/or SUCD4,LDH_D, PFLi, PRO1z, THD2 and/or 7.39974 GLUDy 448 ADHEr, GLUDy, LDH_D,PFLi, PPCK, THD2 and/or GLUDy 7.39109 449 ADHEr, FUM, LDH_D, NADH6,PFLi, THD2 and/or GLUDy 7.3905 450 ADHEr, ASPT, GLCpts, LDH_D, MDH, PPCK7.38004 451 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, NADH6, PGI 7.37787452 ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH, THD2 and/or 7.35321 GLUDy453 ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6 7.31719 454 ADHEr, GLCpts,LDH_D, MDH, PFLi, THD2 and/or GLUDy 7.31585 455 ADHEr, ASPT, LDH_D, MDH,PYK, THD2 and/or GLUDy 7.30613 456 ADHEr, HEX1, LDH_D, ME2, PGL and/orG6PDHy, THD2 and/or 7.30328 GLUDy 457 ADHEr, ASPT, LDH_D, MDH, PPCK,THD2 and/or GLUDy 7.2931 458 ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK7.27491 459 ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK 7.27491 460 ADHEr,LDH_D, ME2, NADH6, PFLi, THD2 and/or GLUDy 7.26202 461 ADHEr, FRD and/orSUCD4, FUM, LDH_D, ME2, PFLi 7.25565 462 ADHEr, LDH_D, MDH, NADH12,NADH6, PFLi 7.25565 463 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PFLi, THD2and/or 7.23622 GLUDy 464 ADHEr, ASPT, LDH_D, MDH, NADH6, THD2 and/orGLUDy 7.23393 465 ADHEr, HEX1, LDH_D, NADH6, PFLi, THD2 and/or GLUDy7.21989 466 ADHEr, FRD and/or SUCD4, FUM, HEX1, LDH_D, PFLi 7.2125 467ADHEr, FRD and/or SUCD4, FUM, LDH_D, PFLi, THD2 and/or 7.21135 GLUDy 468ADHEr, ATPS4r, HEX1, LDH_D, PFLi, THD2 and/or GLUDy 7.20895 469 ADHEr,ATPS4r, LDH_D, MDH, NADH6, THD2 and/or GLUDy 7.19914 470 ADHEr, FRDand/or SUCD4, GLCpts, LDH_D, ME2, PFLi 7.19295 471 ADHEr, ATPS4r, FUM,LDH_D, NADH6, PPCK 7.19078 472 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK7.19078 473 ADHEr, ASPT, GLCpts, LDH_D, MDH, THD2 and/or GLUDy 7.18851474 ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi 7.1878 475 ADHEr, GLUDy,HEX1, LDH_D, PFLi, THD2 and/or GLUDy 7.13398 476 ADHEr, ATPS4r, FUM,LDH_D, ME2, NADH6 7.09625 477 ADHEr, ATPS4r, FUM, HEX1, LDH_D, NADH67.09284 478 ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi 7.06106 479 ADHEr,LDH_D, ME2, NADH12, NADH6, PFLi 7.04349 480 ADHEr, FUM, LDH_D, ME2,NADH6, PFLi 7.03739 481 ADHEr, GLCpts, LDH_D, NADH6, PFLi, THD2 and/orGLUDy 7.02149 482 ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi 7.00666 483ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PPCK 7.00602 484 ADHEr, FUM, HEX1,LDH_D, NADH6, PFLi 7.00047

TABLE 11 Growth-coupled production designs for the 4-HB-CoA:MAA pathway(FIG. 6). Predicted Design MAA ID Metabolic Transformations Targeted forRemoval Yield 1 ATPS4r 1.41919 2 ADHEr, NADH6 5.51519 3 ADHEr, PPCK4.28804 4 ADHEr, FRD and/or SUCD4 4.21382 5 PFLi, PGI 2.45706 6 ATPS4r,THD2 and/or GLUDy 1.92124 7 ADHEr, PGI 1.8023 8 ADHEr, FUM 1.29828 9 FRDand/or SUCD4, PFLi 1.06442 10 NADH6, PFLi 1.06442 11 PFLi, PPCK 0.9393112 ADHEr, HEX1 0.80948 13 ADHEr, PFK and/or FBA and/or TPI 0.32133 14ADHEr, HEX1, PGI 8.71082 15 HEX1, PFLi, PGI 7.22213 16 ADHEr, NADH6,PFLi 6.8792 17 ADHEr, NADH6, PGI 6.18566 18 ADHEr, NADH6, PFK and/or FBAand/or TPI 6.09754 19 ADHEr, MDH, THD2 and/or GLUDy 6.05682 20 ADHEr,NADH6, PPCK 5.89771 21 ADHEr, MDH, NADH6 5.71411 22 ADHEr, NADH6, THD2and/or GLUDy 5.63485 23 ADHEr, FUM, NADH6 5.58019 24 ATPS4r, HEX1, PFLi5.2123 25 ADHEr, PFLi, PPCK 5.15556 26 ATPS4r, PFLi, PGI 5.00885 27ADHEr, PPCK, THD2 and/or GLUDy 4.91748 28 ATPS4r, PFK and/or FBA and/orTPI, PFLi 4.90696 29 ADHEr, PGI, PPCK 4.71025 30 ADHEr, FRD and/orSUCD4, PGI 4.67759 31 ADHEr, PFK and/or FBA and/or TPI, PPCK 4.65664 32ADHEr, HEX1, PFK and/or FBA and/or TPI 4.62311 33 ADHEr, FRD and/orSUCD4, PFK and/or FBA and/or TPI 4.61796 34 ATPS4r, ME2, THD2 and/orGLUDy 4.44391 35 ADHEr, FRD and/or SUCD4, MDH 4.39382 36 ADHEr, GLCpts,PPCK 4.3754 37 ADHEr, FUM, PPCK 4.36046 38 ADHEr, MDH, PPCK 4.36046 39ADHEr, FRD and/or SUCD4, ME2 4.31642 40 ADHEr, FUM, THD2 and/or GLUDy4.30511 41 ADHEr, FRD and/or SUCD4, THD2 and/or GLUDy 4.29689 42 ADHEr,FRD and/or SUCD4, GLCpts 4.27312 43 ADHEr, FUM, HEX1 4.11519 44 ACKrand/or PTAr, AKGD, ATPS4r 3.45333 45 ME2, PGL and/or G6PDHy, THD2 and/orGLUDy 3.397 46 ACKr and/or PTAr, ATPS4r, SUCOAS 3.23462 47 ADHEr, HEX1,THD2 and/or GLUDy 3.09661 48 MDH, PFLi, THD2 and/or GLUDy 3.00855 49ATPS4r, PPCK, PYK 2.7407 50 PFLi, PPCK, THD2 and/or GLUDy 1.87744 51ACKr and/or PTAr, FRD and/or SUCD4, PFLi 1.17455 52 ACKr and/or PTAr,NADH6, PFLi 1.17455 53 MDH, PGL and/or G6PDHy, THD2 and/or GLUDy 1.0705654 ACKr and/or PTAr, PFLi, PPCK 1.03613 55 FUM, PFLi, THD2 and/or GLUDy0.95467 56 ADHEr, ASPT, MDH 0.77578 57 FUM, HEX1, PFLi 0.70584 58 HEX1,PFK and/or FBA and/or TPI, PFLi 0.51097 59 HEX1, PFLi, THD2 and/or GLUDy0.43064 60 ASPT, FUM, PFLi 0.26432 61 ASPT, MDH, PFLi 0.22676 62 ADHEr,MDH, PGL and/or G6PDHy, THD2 and/or GLUDy 9.4382 63 ADHEr, ATPS4r,GLCpts, NADH6 8.91415 64 ADHEr, ATPS4r, NADH6, PGI 7.77864 65 ADHEr, FRDand/or SUCD4, PFLi, PGI 7.64241 66 ADHEr, NADH6, PFLi, PGI 7.64241 67ADHEr, FRD and/or SUCD4, PFK and/or FBA and/or TPI, PFLi 7.61537 68ADHEr, NADH6, PFK and/or FBA and/or TPI, PFLi 7.61537 69 ACKr and/orPTAr, HEX1, PFLi, PGI 7.28181 70 ADHEr, ATPS4r, MDH, NADH6 7.05818 71ADHEr, ASPT, LDH_D, MDH 7.03701 72 ADHEr, NADH6, PFLi, THD2 and/or GLUDy6.99958 73 ADHEr, FRD and/or SUCD4, LDH_D, PFLi 6.98499 74 ADHEr, MDH,NADH6, THD2 and/or GLUDy 6.81498 75 ADHEr, PFLi, PPCK, THD2 and/or GLUDy6.6221 76 ADHEr, NADH6, PPCK, THD2 and/or GLUDy 6.57996 77 ADHEr,ATPS4r, NADH6, PPCK 6.53047 78 ADHEr, HEX1, NADH6, PFK and/or FBA and/orTPI 6.36651 79 ADHEr, ATPS4r, HEX1, NADH6 6.24352 80 ADHEr, NADH6, PGI,THD2 and/or GLUDy 6.23747 81 ADHEr, FRD and/or SUCD4, ME2, THD2 and/orGLUDy 6.22745 82 ADHEr, ME2, NADH6, THD2 and/or GLUDy 6.21872 83 ADHEr,NADH6, PFK and/or FBA and/or TPI, THD2 and/or 6.15963 GLUDy 84 ADHEr,ATPS4r, MDH, THD2 and/or GLUDy 6.13586 85 ADHEr, FUM, ME2, THD2 and/orGLUDy 6.05682 86 ADHEr, GLCpts, NADH6, PPCK 6.01786 87 ADHEr, FUM, PFLi,THD2 and/or GLUDy 5.92596 88 ADHEr, ATPS4r, MDH, PPCK 5.89299 89 ADHEr,ATPS4r, FUM, PPCK 5.89299 90 ADHEr, ASPT, MDH, PYK 5.87822 91 ATPS4r,FUM, PFLi, THD2 and/or GLUDy 5.87538 92 ADHEr, GLCpts, MDH, NADH65.83728 93 ADHEr, FRD and/or SUCD4, GLCpts, PFLi 5.82636 94 ATPS4r, ME2,PGL and/or G6PDHy, THD2 and/or GLUDy 5.77678 95 ADHEr, FUM, ME2, NADH65.71411 96 ADHEr, ATPS4r, FUM, HEX1 5.67609 97 ADHEr, FUM, HEX1, NADH65.6596 98 ADHEr, HEX1, NADH6, THD2 and/or GLUDy 5.56588 99 ME2, PFLi,PGL and/or G6PDHy, THD2 and/or GLUDy 5.37063 100 ADHEr, HEX1, PFLi, PPS5.23221 101 ADHEr, FUM, HEX1, PFLi 5.20022 102 ADHEr, HEX1, MDH, PFLi5.20022 103 ATPS4r, PFLi, PPCK, PYK 5.11897 104 ADHEr, ATPS4r, HEX1, MDH4.91648 105 ADHEr, FUM, PFK and/or FBA and/or TPI, PFLi 4.88964 106ADHEr, MDH, PFK and/or FBA and/or TPI, PFLi 4.88964 107 ADHEr, PFKand/or FBA and/or TPI, PFLi, PPS 4.88619 108 ADHEr, FRD and/or SUCD4,HEX1, PFK and/or FBA and/or TPI 4.77488 109 ADHEr, FUM, PGI, THD2 and/orGLUDy 4.72683 110 ADHEr, MDH, PGI, THD2 and/or GLUDy 4.72683 111 ADHEr,FUM, PFK and/or FBA and/or TPI, THD2 and/or GLUDy 4.67659 112 ADHEr,MDH, PFK and/or FBA and/or TPI, THD2 and/or GLUDy 4.67659 113 ADHEr, FRDand/or SUCD4, PPCK, PYK 4.5823 114 ADHEr, FRD and/or SUCD4, LDH_D, PPCK4.50446 115 ADHEr, FRD and/or SUCD4, GLCpts, MDH 4.48854 116 ADHEr,GLCpts, MDH, THD2 and/or GLUDy 4.48038 117 ADHEr, MDH, PYK, THD2 and/orGLUDy 4.46392 118 ADHEr, GLCpts, MDH, PPCK 4.44991 119 ADHEr, FUM,GLCpts, PPCK 4.44991 120 ADHEr, GLCpts, PPCK, THD2 and/or GLUDy 4.43743121 ADHEr, MDH, PPCK, PYK 4.43142 122 ADHEr, FUM, PPCK, PYK 4.43142 123ADHEr, FRD and/or SUCD4, GLCpts, ME2 4.40879 124 ADHEr, FRD and/orSUCD4, FUM, ME2 4.39382 125 ADHEr, FUM, GLCpts, THD2 and/or GLUDy 4.3649126 ADHEr, FRD and/or SUCD4, GLCpts, THD2 and/or GLUDy 4.3533 127 ME2,NADH6, PGL and/or G6PDHy, THD2 and/or GLUDy 4.08146 128 ADHEr, HEX1, PGLand/or G6PDHy, THD2 and/or GLUDy 4.06156 129 ADHEr, ATPS4r, FUM, PGLand/or G6PDHy 3.76615 130 ADHEr, ATPS4r, HEX1, THD2 and/or GLUDy 3.73541131 ACKr and/or PTAr, AKGD, ATPS4r, THD2 and/or GLUDy 3.65142 132 ACKrand/or PTAr, ME2, PGL and/or G6PDHy, THD2 and/or 3.52563 GLUDy 133ADHEr, ASPT, ATPS4r, MDH 3.50899 134 ACKr and/or PTAr, ATPS4r, SUCOAS,THD2 and/or GLUDy 3.4624 135 ACKr and/or PTAr, ATPS4r, PFK and/or FBAand/or TPI, SUCOAS 3.3813 136 ATPS4r, PPCK, PYK, THD2 and/or GLUDy3.23342 137 ACKr and/or PTAr, MDH, PFLi, THD2 and/or GLUDy 3.16308 138ATPS4r, NADH6, PDH, PFLi 3.01078 139 FUM, ME2, PFLi, THD2 and/or GLUDy3.00855 140 AKGD, MDH, PGL and/or G6PDHy, THD2 and/or GLUDy 2.70882 141MDH, PGL and/or G6PDHy, SUCOAS, THD2 and/or GLUDy 2.58524 142 ACKrand/or PTAr, GLU5K, PFLi, PGI 2.51808 143 ACKr and/or PTAr, G5SD, PFLi,PGI 2.51808 144 ATPS4r, MDH, PGL and/or G6PDHy, THD2 and/or GLUDy2.13925 145 ME2, NADH6, PFLi, THD2 and/or GLUDy 1.97605 146 ACKr and/orPTAr, PFLi, PPCK, THD2 and/or GLUDy 1.96877 147 FUM, MDH, PGL and/orG6PDHy, THD2 and/or GLUDy 1.58766 148 ADHEr, ATPS4r, HEX1, PPS 1.57755149 ACKr and/or PTAr, MDH, PGL and/or G6PDHy, THD2 and/or 1.20253 GLUDy150 ACKr and/or PTAr, FUM, PFLi, THD2 and/or GLUDy 1.05304 151 ACKrand/or PTAr, ADHEr, ASPT, MDH 0.90077 152 ACKr and/or PTAr, FUM, HEX1,PFLi 0.88292 153 ACKr and/or PTAr, HEX1, PFLi, THD2 and/or GLUDy 0.61521154 ACKr and/or PTAr, HEX1, PFK and/or FBA and/or TPI, PFLi 0.56359 155ASPT, FUM, PDH, PFLi 0.43285 156 ASPT, MDH, PDH, PFLi 0.39171 157 ADHEr,ASPT, ATPS4r, GLCpts, MDH 13.10515 158 ACKr and/or PTAr, ADHEr, LDH_D,MDH, NADH6 11.66583 159 ADHEr, ATPS4r, MDH, PGL and/or G6PDHy, THD2and/or GLUDy 11.54962 160 ATPS4r, MDH, PDH, PGL and/or G6PDHy, THD2and/or GLUDy 11.18163 161 ADHEr, LDH_D, MDH, PGL and/or G6PDHy, THD2and/or GLUDy 10.87211 162 ADHEr, ASPT, MDH, PGL and/or G6PDHy, PYK10.85118 163 ADHEr, FUM, ME2, PGL and/or G6PDHy, THD2 and/or GLUDy10.79537 164 ADHEr, MALS, MDH, PGL and/or G6PDHy, THD2 and/or GLUDy10.79537 165 ADHEr, ICL, MDH, PGL and/or G6PDHy, THD2 and/or GLUDy10.79537 166 ADHEr, ASPT, ATPS4r, LDH_D, MDH 10.36931 167 ATPS4r,GLCpts, NADH6, PDH, PFLi 10.18817 168 ADHEr, ATPS4r, GLCpts, NADH6, PFLi10.10757 169 ACKr and/or PTAr, ADHEr, MDH, PGL and/or G6PDHy, THD29.4975 and/or GLUDy 170 ADHEr, FRD and/or SUCD4, ME2, PGL and/or G6PDHy,THD2 9.20833 and/or GLUDy 171 ADHEr, ATPS4r, LDH_D, NADH6, PFLi 9.04248172 ADHEr, GLCpts, MDH, NADH6, THD2 and/or GLUDy 8.60394 173 ADHEr, ME2,NADH6, PGL and/or G6PDHy, THD2 and/or GLUDy 8.57189 174 ADHEr, ATPS4r,LDH_D, MDH, NADH6 8.07655 175 FUM, MDH, PGL and/or G6PDHy, PYK, THD2and/or GLUDy 8.03861 176 MDH, ME2, PGL and/or G6PDHy, PYK, THD2 and/orGLUDy 8.03861 177 ADHEr, FRD and/or SUCD4, LDH_D, PFLi, PPCK 7.63009 178ADHEr, ATPS4r, FUM, LDH_D, NADH6 7.6151 179 ADHEr, ASPT, MDH, PYK, THD2and/or GLUDy 7.44202 180 ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH7.40564 181 ADHEr, LDH_D, NADH6, PFLi, PPCK 7.40192 182 ADHEr, FRDand/or SUCD4, LDH_D, MDH, PFLi 7.36994 183 ADHEr, ASPT, ATPS4r, MDH, PGLand/or G6PDHy 7.36609 184 ADHEr, ASPT, LDH_D, MDH, PPCK 7.33413 185ADHEr, LDH_D, MDH, PFLi, THD2 and/or GLUDy 7.27861 186 ADHEr, NADH6,PFLi, PPCK, PYK 7.27816 187 ADHEr, ASPT, LDH_D, MDH, NADH6 7.2681 188ADHEr, FUM, LDH_D, PFLi, PPCK 7.23481 189 ADHEr, LDH_D, MDH, PFLi, PPCK7.23481 190 ADHEr, ASPT, GLCpts, LDH_D, MDH 7.21534 191 ADHEr, FRDand/or SUCD4, FUM, LDH_D, PFLi 7.16964 192 ADHEr, FRD and/or SUCD4,LDH_D, ME2, PFLi 7.15036 193 ADHEr, LDH_D, MDH, NADH6, PFLi 7.14486 194ADHEr, ASPT, LDH_D, MDH, THD2 and/or GLUDy 7.14394 195 ADHEr, FUM,LDH_D, PFLi, THD2 and/or GLUDy 7.13176 196 ASPT, ATPS4r, MDH, PGL and/orG6PDHy, PYK 7.127 197 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PFLi 7.11053198 ADHEr, FRD and/or SUCD4, LDH_D, PFLi, THD2 and/or GLUDy 7.09551 199ADHEr, ATPS4r, FUM, ME2, NADH6 7.05818 200 ATPS4r, HEX1, ME2, PFLi, THD2and/or GLUDy 7.02076 201 ADHEr, ATPS4r, NADH6, PGL and/or G6PDHy, PPCK6.9884 202 ADHEr, LDH_D, NADH12, NADH6, PFLi 6.98499 203 ADHEr, FUM,LDH_D, NADH6, PFLi 6.98167 204 ATPS4r, PFLi, PPCK, PYK, THD2 and/orGLUDy 6.96842 205 ADHEr, HEX1, PFLi, PPS, THD2 and/or GLUDy 6.92886 206ADHEr, HEX1, ME2, PGL and/or G6PDHy, THD2 and/or GLUDy 6.88098 207ADHEr, FUM, ME2, NADH6, THD2 and/or GLUDy 6.81498 208 ADHEr, ATPS4r,HEX1, NADH6, PGL and/or G6PDHy 6.72128 209 ATPS4r, FUM, NADH12, PFLi,THD2 and/or GLUDy 6.67745 210 ADHEr, ME2, PGL and/or G6PDHy, PPCK, THD2and/or GLUDy 6.67685 211 ADHEr, ATPS4r, HEX1, MDH, PFLi 6.56359 212ADHEr, ATPS4r, HEX1, NADH6, PFK and/or FBA and/or TPI 6.49377 213 ADHEr,HEX1, MDH, PFLi, THD2 and/or GLUDy 6.48868 214 ADHEr, GLCpts, NADH6,PGI, PPCK 6.48565 215 ADHEr, GLCpts, NADH6, PFK and/or FBA and/or TPI,PPCK 6.48238 216 ADHEr, ATPS4r 1.41919 217 ADHEr, PFLi, PGI 2.45706 218ADHEr, ATPS4r, THD2 and/or GLUDy 1.92124 219 ADHEr, ATPS4r, HEX1, PFLi5.2123 220 ADHEr, ATPS4r, PFLi, PGI 5.00885 221 ADHEr, ATPS4r, PFKand/or FBA and/or TPI, PFLi 4.90696 222 ADHEr, ATPS4r, ME2, THD2 and/orGLUDy 4.44391 223 ACKr and/or PTAr, ADHEr, AKGD, ATPS4r 3.45333 224ADHEr, ME2, PGL and/or G6PDHy, THD2 and/or GLUDy 3.397 225 ACKr and/orPTAr, ADHEr, ATPS4r, SUCOAS 3.23462 226 ADHEr, ATPS4r, PPCK, PYK 2.7407227 ADHEr, ATPS4r, HEX1, PFLi, PGI 6.94738 228 ADHEr, ATPS4r, FUM, PFLi,THD2 and/or GLUDy 5.87538 229 ADHEr, ATPS4r, ME2, PGL and/or G6PDHy,THD2 and/or GLUDy 5.77678 230 ADHEr, ME2, PFLi, PGL and/or G6PDHy, THD2and/or GLUDy 5.37063 231 ADHEr, ATPS4r, PFLi, PPCK, PYK 5.11897 232ADHEr, ATPS4r, MDH, PDH, PGL and/or G6PDHy, THD2 and/or 11.18163 GLUDy233 ADHEr, ATPS4r, GLCpts, NADH6, PDH, PFLi 10.18817 234 ADHEr, ATPS4r,HEX1, ME2, PFLi, THD2 and/or GLUDy 7.02076 235 ADHEr, ATPS4r, PFLi,PPCK, PYK, THD2 and/or GLUDy 6.96842 236 ADHEr, ATPS4r, FUM, NADH12,PFLi, THD2 and/or GLUDy 6.67745 237 ADHEr, ATPS4r, MDH, PFK and/or FBAand/or TPI, PFLi, THD2 6.00293 and/or GLUDy 238 ADHEr, LDH_D, MDH, THD2and/or GLUDy 6.05682 239 ADHEr, LDH_D, MDH, NADH6 5.71411 240 ADHEr,LDH_D, MDH, PPCK 4.36046 241 ADHEr, LDH_D, MDH, PGI 1.8023 242 ADHEr,HEX1, LDH_D, MDH 0.71076 243 ADHEr, HEX1, LDH_D, MDH, PGI 8.71082 244ADHEr, LDH_D, MDH, NADH6, THD2 and/or GLUDy 6.81498 245 ACKr and/orPTAr, ADHEr, LDH_D, MDH, THD2 and/or GLUDy 6.26208 246 ADHEr, GLUDy,LDH_D, MDH, THD2 and/or GLUDy 6.1932 247 ADHEr, LDH_D, MDH, PPCK, THD2and/or GLUDy 6.18632 248 ADHEr, LDH_D, MDH, NADH6, PGI 6.18566 249ADHEr, ATPS4r, LDH_D, MDH, THD2 and/or GLUDy 6.13586 250 ADHEr, ATPS4r,LDH_D, MDH, PPCK 5.89299 251 ADHEr, GLCpts, LDH_D, MDH, NADH6 5.83728252 ADHEr, HEX1, LDH_D, MDH, PFLi 5.20022 253 ADHEr, ASPT, ATPS4r,GLCpts, LDH_D, MDH 13.10515 254 ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH613.09985 255 ADHEr, ASPT, LDH_D, MDH, PFLi, THD2 and/or GLUDy 12.43893256 ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, THD2 and/or 12.0485GLUDy 257 ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, THD2 and/or11.54962 GLUDy 258 ADHEr, ASPT, ATPS4r, LDH_D, MDH, PFLi 10.9713 259ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy 10.87514 260 ADHEr,ASPT, LDH_D, MDH, PGL and/or G6PDHy, PYK 10.85118 261 ADHEr, ASPT,LDH_D, MDH, PFLi, PYK 10.69852 262 ADHEr, ASPT, ATPS4r, LDH_D, MDH, PPCK10.54348 263 ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6 9.24298 264 ADHEr,ATPS4r, GLCpts, LDH_D, MDH, NADH6 8.89824 265 ADHEr, GLUDy, LDH_D, MDH,PFLi, THD2 and/or GLUDy 8.78148 266 ADHEr, LDH_D, MDH, PFLi, PPCK, THD2and/or GLUDy 8.75525 267 ADHEr, LDH_D, MDH, NADH6, PFLi, THD2 and/orGLUDy 8.68453 268 ADHEr, GLCpts, LDH_D, MDH, NADH6, THD2 and/or GLUDy8.60394 269 ADHEr, ATPS4r, LDH_D, MDH, PFLi, THD2 and/or GLUDy 8.54512270 ADHEr, HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy 8.41994 271 ADHEr,ATPS4r, LDH_D, MDH, NADH6, THD2 and/or GLUDy 8.22075 272 ADHEr, LDH_D,MDH, NADH12, NADH6, THD2 and/or GLUDy 8.20953 273 ADHEr, ASPT, LDH_D,MDH, PGI, THD2 and/or GLUDy 7.83287 274 ACKr and/or PTAr, ADHEr, LDH_D,MDH, PPCK, THD2 and/or 7.76113 GLUDy 275 ADHEr, LDH_D, MDH, PFLi, PGI,THD2 and/or GLUDy 7.73026 276 ADHEr, LDH_D, MDH, NADH6, PFLi, PGI7.64241 277 ADHEr, LDH_D, MDH, NADH6, PFLi, PPCK 7.62766 278 ADHEr,ASPT, LDH_D, MDH, NADH6, PPCK 7.5687 279 ADHEr, ASPT, GLCpts, LDH_D,MDH, PPCK 7.50936 280 ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6 7.45108 281ADHEr, ASPT, LDH_D, MDH, PYK, THD2 and/or GLUDy 7.44202 282 ADHEr,GLCpts, LDH_D, MDH, PFLi, THD2 and/or GLUDy 7.41734 283 ADHEr, ASPT,LDH_D, MDH, PPCK, THD2 and/or GLUDy 7.40692 284 ADHEr, GLCpts, LDH_D,MDH, PFLi, PPCK 7.38196 285 ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi7.36994 286 ADHEr, ASPT, LDH_D, MDH, NADH6, THD2 and/or GLUDy 7.35125287 ADHEr, ATPS4r, LDH_D, MDH, PFLi, PPCK 7.34743 288 ADHEr, ASPT,GLCpts, LDH_D, MDH, THD2 and/or GLUDy 7.30247 289 ADHEr, GLCpts, LDH_D,MDH, NADH6, PFLi 7.29756 290 ADHEr, ASPT, GLUDy, LDH_D, MDH, THD2 and/orGLUDy 7.20317 291 ACKr and/or PTAr, ADHEr, GLUDy, LDH_D, MDH, THD2and/or 6.86403 GLUDy 292 ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi 6.56359293 ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, THD2 and/or 6.38107GLUDy 294 ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, PPS 6.33965 295ADHEr, LDH_D, MDH, NADH6, PYK, THD2 and/or GLUDy 6.13919 296 ADHEr,LDH_D, MDH, NADH6, PPCK, PYK 6.10615 297 ADHEr, LDH_D, MDH, NADH12,NADH6, PPCK 6.03902 298 ADHEr, ATPS4r, LDH_D, MDH, PPCK, THD2 and/orGLUDy 5.95979 299 ADHEr, ATPS4r, LDH_D, MDH, PFLi, PGI 5.00885 300 NA0.40409 301 FRD and/or SUCD4 0.34622 302 ADHEr, ATPS4r, NADH6 0.29582303 ADHEr, ATPS4r, PPCK 0.24649 304 ADHEr, NADH12, NADH6 0.28919 305ADHEr, FRD and/or SUCD4, LDH_D 0.26599 306 MDH, NADH6, PFLi 0.27181 307FRD and/or SUCD4, ME2, PFLi 0.27027 308 FRD and/or SUCD4, MDH, PFLi0.26589 309 FUM, PFLi, PPCK 0.25998 310 MDH, PFLi, PPCK 0.25998 311NADH6, PFLi, PPCK 0.25452 312 FRD and/or SUCD4, PFLi, PPCK 0.24933 313ATPS4r, PFLi, PPCK 0.2281 314 FRD and/or SUCD4, FUM, PFLi 0.30093 315PFK and/or FBA and/or TPI, PFLi, PPCK 0.14537 316 PFLi, PGI, PPCK0.14284 317 ADHEr, ATPS4r, PGI 0.23063 318 NADH12, NADH6, PFLi 0.30419319 FUM, NADH6, PFLi 0.30486 320 ADHEr, FRD and/or SUCD4, PPCK 0.21424321 ADHEr, ATPS4r, PFK and/or FBA and/or TPI 0.23674 322 ADHEr, ATPS4r,HEX1 0.37576 323 ADHEr, MDH, PGI 0.24821 324 ADHEr, FUM, PGI 0.24821 325FUM, HEX1, PFK and/or FBA and/or TPI 0.18252 326 ADHEr, ATPS4r, HEX1,PGI 0.08649 327 ADHEr, ATPS4r, HEX1, PFK and/or FBA and/or TPI 0.08878328 ADHEr, LDH_D, NADH6, PPCK 0.17351 329 ADHEr, FRD and/or SUCD4,LDH_D, MDH 0.18263 330 ATPS4r, MDH, PDH, PGL and/or G6PDHy 0.19729 331ADHEr, ATPS4r, PPCK, THD2 and/or GLUDy 0.21063 332 ADHEr, FRD and/orSUCD4, FUM, LDH_D 0.23871 333 ADHEr, FRD and/or SUCD4, HEX1, LDH_D0.24228 334 ADHEr, ME2, NADH12, NADH6 0.27452 335 ADHEr, ATPS4r, MDH,PGL and/or G6PDHy 0.26541 336 ADHEr, ATPS4r, NADH6, PFK and/or FBAand/or TPI 0.11424 337 ADHEr, ATPS4r, ME2, NADH6 0.28029 338 ADHEr,ATPS4r, NADH12, NADH6 0.28705 339 ACKr and/or PTAr, AKGD, ATPS4r, PFKand/or FBA and/or TPI 0.10833 340 ADHEr, ATPS4r, FUM, THD2 and/or GLUDy0.23122 341 ADHEr, HEX1, NADH12, NADH6 0.2788 342 ATPS4r, FDH2, NADH6,PDH 0.16467 343 ATPS4r, GLCpts, MDH, PGL and/or G6PDHy 0.23908 344ADHEr, FUM, LDH_D, THD2 and/or GLUDy 0.35439 345 ADHEr, FUM, LDH_D,NADH6 0.26726 346 FUM, ME2, NADH6, PFLi 0.27181 347 ME2, NADH12, NADH6,PFLi 0.27027 348 FRD and/or SUCD4, FUM, PFLi, THD2 and/or GLUDy 0.2584349 FUM, NADH6, PFLi, THD2 and/or GLUDy 0.2584 350 FRD and/or SUCD4,PFLi, PRO1z, THD2 and/or GLUDy 0.25563 351 ADHEr, HEX1, LDH_D, PPS0.37122 352 FUM, NADH12, NADH6, PFLi 0.30093 353 ADHEr, FUM, PFLi, PGI0.2381 354 ADHEr, MDH, PFLi, PGI 0.2381 355 ASPT, ATPS4r, FUM, PDH0.19443 356 ASPT, ATPS4r, MDH, PDH 0.18797 357 ADHEr, ASPT, MDH, THD2and/or GLUDy 0.20167 358 FUM, HEX1, PFLi, THD2 and/or GLUDy 0.32948 359ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, MDH 0.22488 360 ADHEr, HEX1,LDH_D, NADH6 0.28496 361 ACKr and/or PTAr, ADHEr, NADH6, PPCK 0.21426362 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, PPCK 0.2103 363 FBP,PFLi, PGI, PPCK 0.14228 364 PFK and/or FBA and/or TPI, PFLi, PGI, PPCK0.14228 365 ADHEr, HEX1, LDH_D, PPCK 0.26035 366 MDH, PDH, PFLi, PGI0.21218 367 FUM, PDH, PFLi, PGI 0.21218 368 ADHEr, ASPT, ATPS4r, FUM0.22058 369 HEX1, NADH6, PFK and/or FBA and/or TPI, PFLi 0.1381 370 FUM,PFK and/or FBA and/or TPI, PFLi, THD2 and/or GLUDy 0.13758 371 FUM,PFLi, PGI, THD2 and/or GLUDy 0.1353 372 HEX1, NADH6, PFLi, PGI 0.13506373 ADHEr, FUM, LDH_D, PPCK 0.20562 374 ATPS4r, GLCpts, NADH6, PFLi0.26825 375 ADHEr, FRD and/or SUCD4, FUM, THD2 and/or GLUDy 0.2204 376ADHEr, FUM, NADH6, THD2 and/or GLUDy 0.2204 377 ADHEr, FRD and/or SUCD4,PRO1z, THD2 and/or GLUDy 0.21775 378 ATPS4r, HEX1, PFLi, PGI 0.08042 379ATPS4r, HEX1, PFK and/or FBA and/or TPI, PFLi 0.0824 380 ASPT, ATPS4r,GLCpts, MDH 0.25562 381 ATPS4r, GLCpts, MDH, PPCK 0.24489 382 ATPS4r,FUM, GLCpts, PPCK 0.24489 383 ADHEr, ASPT, FUM, ME2 0.22754 384 ADHEr,ASPT, FUM, THD2 and/or GLUDy 0.22026 385 ADHEr, FRD and/or SUCD4, HEX1,PGI 0.08245 386 ADHEr, HEX1, NADH6, PGI 0.08245 387 ADHEr, ASPT, FUM,LDH_D 0.2051 388 ATPS4r, GLCpts, MDH, THD2 and/or GLUDy 0.23421 389ADHEr, HEX1, PFK and/or FBA and/or TPI, PPCK 0.08078 390 ADHEr, MDH,ME2, NADH6 0.23176 391 ADHEr, MDH, PFLi, THD2 and/or GLUDy 2.6304 392ADHEr, FRD and/or SUCD4, FUM, PFLi 0.37735 393 ADHEr, HEX1, PGI, PPCK0.13376 394 ADHEr, FRD and/or SUCD4, HEX1, PFLi 0.03691 395 ADHEr,ATPS4r, GLCpts, MDH, PDH, PGL and/or G6PDHy 9.03958 396 ADHEr, ATPS4r,LDH_D, NADH12, NADH6, PFLi 9.0297 397 ADHEr, ATPS4r, LDH_D, MDH, NADH6,PGL and/or G6PDHy 8.92362 398 ACKr and/or PTAr, ADHEr, ATPS4r, MDH,NADH6, PGL and/or 8.83429 G6PDHy 399 ACKr and/or PTAr, ADHEr, ATPS4r,GLCpts, MDH, PGL and/or 8.62906 G6PDHy 400 ADHEr, MDH, ME2, PGL and/orG6PDHy, PYK, THD2 and/or 8.50911 GLUDy 401 ADHEr, FUM, MDH, PGL and/orG6PDHy, PYK, THD2 and/or 8.50911 GLUDy 402 ACKr and/or PTAr, ADHEr, FRDand/or 8.37268 SUCD4, LDH_D, MDH, THD2 and/or GLUDy 403 ADHEr, ATPS4r,GLCpts, MDH, PGL and/or G6PDHy, THD2 and/or 8.32112 GLUDy 404 ADHEr,ATPS4r, GLCpts, MDH, PGL and/or G6PDHy, PPCK 7.82056 405 ADHEr, FRDand/or SUCD4, HEX1, LDH_D, PFLi, THD2 and/or 7.65378 GLUDy 406 ACKrand/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, MDH, PGI 7.4765 407 ADHEr,ME2, NADH12, NADH6, PGL and/or G6PDHy, THD2 7.16841 and/or GLUDy 408ADHEr, HEX1, LDH_D, PFLi, PPS, THD2 and/or GLUDy 6.91902 409 ACKr and/orPTAr, ADHEr, ATPS4r, GLCpts, NADH6, PGI 6.85613 410 ADHEr, ATPS4r, FUM,LDH_D, NADH6, PPCK 6.78808 411 ADHEr, ATPS4r, FUM, LDH_D, ME2, NADH66.71695 412 ADHEr, ASPT, ATPS4r, GLCpts, MDH, PGL and/or G6PDHy 6.67975413 ACKr and/or PTAr, ADHEr, FRD and/or 6.31121 SUCD4, LDH_D, PPCK, THD2and/or GLUDy 414 ADHEr, ATPS4r, ME2, PGL and/or G6PDHy, PPCK, THD2and/or 6.23672 GLUDy 415 ACKr and/or PTAr, ADHEr, FUM, LDH_D, ME2, NADH66.19739 416 ADHEr, ATPS4r, FUM, LDH_D, NADH6, THD2 and/or GLUDy 6.15859417 ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, NADH6 6.06031 418 ADHEr,ATPS4r, FUM, HEX1, LDH_D, NADH6 5.95481 419 ACKr and/or PTAr, ADHEr,HEX1, LDH_D, NADH6, THD2 and/or 5.89694 GLUDy 420 ADHEr, FUM, HEX1,LDH_D, PPS, THD2 and/or GLUDy 5.87873 421 ADHEr, FUM, HEX1, LDH_D,NADH12, NADH6 5.87075 422 ADHEr, ATPS4r, NADH12, NADH6, PRO1z, THD2and/or GLUDy 5.87047 423 ADHEr, FUM, HEX1, LDH_D, ME2, THD2 and/or GLUDy5.85008 424 ACKr and/or PTAr, ADHEr, LDH_D, ME2, NADH12, NADH6 5.77866425 ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, THD2 and/or 5.77062 GLUDy426 ADHEr, HEX1, LDH_D, NADH12, NADH6, THD2 and/or GLUDy 5.74841 427ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PPS, THD2 and/or 5.74605 GLUDy 428ACKr and/or PTAr, ADHEr, CITL, LDH_D, NADH12, NADH6 5.71072 429 ADHEr,ATPS4r, MDH, NADH6, PGL and/or G6PDHy, THD2 and/or 5.70312 GLUDy 430ACKr and/or PTAr, ADHEr, ATPS4r, GLCpts, ME2, NADH6 5.68643 431 ADHEr,ATPS4r, FUM, LDH_D, PFLi, THD2 and/or GLUDy 5.64027 432 ACKr and/orPTAr, ADHEr, CITL, HEX1, LDH_D, NADH6 5.63442 433 ADHEr, FUM, LDH_D,ME2, NADH6, THD2 and/or GLUDy 5.58306 434 ADHEr, ATPS4r, FUM, GLCpts,ME2, PGL and/or G6PDHy 5.57201 435 ADHEr, ATPS4r, HEX1, ME2, PGL and/orG6PDHy, THD2 and/or 5.55702 GLUDy 436 ADHEr, FRD and/or SUCD4, GLUDy,LDH_D, PFLi, THD2 and/or 5.54389 GLUDy 437 ADHEr, FUM, GLUDy, LDH_D,ME2, THD2 and/or GLUDy 5.50066 438 ADHEr, ATPS4r, HEX1, LDH_D, NADH6,THD2 and/or GLUDy 5.47201 439 ADHEr, FUM, GLUDy, LDH_D, PFLi, THD2and/or GLUDy 5.4078 440 ACKr and/or PTAr, ADHEr, ATPS4r, GLCpts, NADH6,PPCK 5.36087 441 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PPCK, THD2 and/or5.34992 GLUDy 442 ADHEr, ATPS4r, FUM, GLCpts, NADH6, PPCK 5.25122 443ACKr and/or PTAr, ADHEr, ATPS4r, CITL, GLCpts, NADH6 5.24209 444 ADHEr,ATPS4r, FUM, LDH_D, PGL and/or G6PDHy, PPCK 5.21165 445 ADHEr, ASPT,ATPS4r, MDH, PDH, PGL and/or G6PDHy 5.10503 446 ADHEr, FUM, HEX1, LDH_D,PFLi, THD2 and/or GLUDy 5.07597 447 ADHEr, FRD and/or SUCD4, HEX1,LDH_D, PFLi, PGI 5.0175 448 ADHEr, ICL, LDH_D, MDH, PGL and/or G6PDHy,THD2 and/or 10.42846 GLUDy 449 ADHEr, LDH_D, MALS, MDH, PGL and/orG6PDHy, THD2 and/or 10.42846 GLUDy 450 ADHEr, GLCpts, LDH_D, MDH, PGLand/or G6PDHy, THD2 and/or 10.30271 GLUDy 451 ADHEr, ATPS4r, LDH_D, MDH,NADH6, PFLi 8.05255 452 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH,NADH6 6.92399 453 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK 6.78808 454ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, NADH6 6.43185 455 ADHEr,ATPS4r, GLCpts, LDH_D, MDH, PGL and/or G6PDHy 5.57201 456 ADHEr, LDH_D,MDH, NADH12, NADH6, PGI 5.02702

TABLE 12 Enzyme names, abbreviations, and the corresponding reactionstoichiometries of designs in Tables 10 and 11. Abbrev. Enzyme NameEquation ABTA 4-aminobutyrate [c]: 4abut + akg --> glu-L + sucsaltransaminase ACKr acetate kinase [c]: ac + atp <==> actp + adp ACSacetyl-CoA synthetase [c]: ac + atp + coa --> accoa + amp + ppi ACt6acetate transport in/out ac[e] + h[e] <==> ac[c] + h[c] via protonsymport ADHEr acetaldehyde-CoA [c]: accoa + (2) h + (2) nadh <==> coa +etoh + (2) dehydrogenase nad AKGD 2-oxoglutarate [c]: akg + coa + nad--> co2 + nadh + succoa dehydrogenase ASNN L-asparaginase [c]: asn-L +h2o --> asp-L + nh4 ASNS1 asparagine synthase [c]: asp-L + atp + gln-L +h2o --> amp + asn-L + glu- (glutamine-hydrolysing) L + h + ppi ASNS2asparagine synthetase [c]: asp-L + atp + nh4 --> amp + asn-L + h + ppiASPT L-aspartase [c]: asp-L --> fum + nh4 ATPS4r ATP synthase (fouradp[c] + (4) h[e] + pi[c] <==> atp[c] + (3) h[c] + protons for one ATP)h2o[c] CBMK2 Carbamate kinase [c]: atp + co2 + nh4 --> adp + cbp + (2) hCITL Citrate lyase [c]: cit --> ac + oaa DAAD D-Amino acid [c]: ala-D +fad + h2o --> fadh2 + nh4 + pyr dehydrogenase EDA 2-dehydro-3-deoxy-[c]: 2ddg6p --> g3p + pyr phosphogluconate aldolase FADH4 FADHdehydrogenaase [c]: fadh2 + mqn8 --> fad + mql8 FBAfructose-bisphosphate [c]: fdp <==> dhap + g3p aldolase FBPfructose-bisphosphatase [c]: fdp + h2o --> f6p + pi FRD fumaratereductase [c]: fum + [electron donor] --> [electron acceptor] + succ FUMfumarase [c]: fum + h2o <==> mal-L G5SD glutamate-5- [c]: glu5p + h +nadph --> glu5sa + nadp + pi semialdehyde dehydrogenase G6PDHy glucose6-phosphate [c]: g6p + nadp <==> 6pgl + h + nadph dehydrogenase G6PDHyglucose 6-phosphate [c]: g6p + nadp <==> 6pgl + h + nadph dehydrogenaseGLCpts D-glucose transport glc-D[e] + pep[c] --> g6p[c] + pyr[c] viaPEP:Pyr PTS GLU5K glutamate 5-kinase [c]: atp + glu-L --> adp + glu5pGLUDC glutamate decarboxylase [c]: glu-L + h --> 4abut + co2 GLUDyglutamate dehydrogenase [c]: glu-L + h2o + nadp <==> akg + h + nadph +nh4 (NADP) GLUDy glutamate dehydrogenase [c]: glu-L + h2o + nadp <==>akg + h + nadph + nh4 (NADP) GLUSy glutamate synthase [c]: akg + gln-L +h + nadph --> (2) glu-L + nadp (NADPH) GLYCL Glycine Cleavage System[c]: gly + nad + thf --> co2 + mlthf + nadh + nh4 HEX1 hexokinase (D-[c]: atp + glc-D --> adp + g6p + h glucose:ATP) ICL Isocitrate lyase[c]: icit --> glx + succ LDH_D D-lactate dehydrogenase [c]: lac-D + nad<==> h + nadh + pyr MALS malate synthase [c]: accoa + glx + h2o -->coa + h + mal-L MDH malate dehydrogenase [c]: mal-L + nad <==> h +nadh + oaa ME1x malic enzyme (NAD) [c]: mal-L + nad --> co2 + nadh + pyrME2 malic enzyme (NADP) [c]: mal-L + nadp --> co2 + nadph + pyr NACODAN-acetylornithine [c]: acg5sa + h2o --> ac + glu5sa deacetylase NADH12NADH dehydrogenase [c]: h + nadh + ubq8 --> nad + ubq8h2 NADH6 NADHdehydrogenase (4.5) h[c] + nadh[c] + ubq8[c] --> (3.5) h[e] + nad[c] +ubq8h2[c] ORNTA ornithine transaminase [c]: akg + orn-L --> glu-L +glu5sa P5CD 1-pyrroline-5-carboxylate [c]: 1pyr5c + (2) h2o + nad -->glu-L + h + nadh dehydrogenase PDH pyruvate dehydrogenase [c]: coa +nad + pyr --> accoa + co2 + nadh PFK phosphofructokinase [c]: atp + f6p--> adp + fdp + h PFLi pyruvate formate lyase [c]: coa + pyr --> accoa +for PGDH phosphogluconate [c]: 6pgc + nadp --> co2 + nadph + ru5p-Ddehydrogenase PGDHY phosphogluconate [c]: 6pgc --> 2ddg6p + h2odehydratase PGI glucose-6-phosphate [c]: g6p <==> f6p isomerase PGL 6-[c]: 6pgl + h2o --> 6pgc + h phosphogluconolactonase PGL 6- [c]: 6pgl +h2o --> 6pgc + h phosphogluconolactonase PGM phosphoglycerate mutase[c]: 3pg <==> 2pg PPCK phosphoenolpyruvate [c]: atp + oaa --> adp +co2 + pep carboxykinase PPS phosphoenolpyruvate [c]: atp + h2o + pyr -->amp + (2) h + pep + pi synthase PRO1z proline oxidase [c]: fad + pro-L--> 1pyr5c + fadh2 + h PTAr phosphotransacetylase [c]: accoa + pi <==>actp + coa PYK pyruvate kinase [c]: adp + h + pep --> atp + pyr RPEribulose 5-phosphate 3- [c]: ru5p-D <==> xu5p-D epimerase SERD_LL-serine deaminase [c]: ser-L --> nh4 + pyr SUCD4 succinatedehyrdogenase [c]: fadh2 + ubq8 <==> fad + ubq8h2 SUCD4 succinatedehyrdogenase [c]: fadh2 + ubq8 <==> fad + ubq8h2 SUCOAS succinyl-CoAsynthetase [c]: atp + coa + succ <==> adp + pi + succoa (ADP-forming)TAL transaldolase [c]: g3p + s7p <==> e4p + f6p THD2 NAD(P) (2) h[e] +nadh[c] + nadp[c] --> (2) h[c] + nad[c] + transhydrogenase nadph[c] THD2NAD(P) (2) h[e] + nadh[c] + nadp[c] --> (2) h[c] + nad[c] +transhydrogenase nadph[c] THD5 NAD transhydrogenase [c]: nad + nadph -->nadh + nadp TKT1 transketolase [c]: r5p + xu5p-D <==> g3p + s7p TKT2transketolase [c]: e4p + xu5p-D <==> f6p + g3p TPI triose-phosphate [c]:dhap <==> g3p isomerase VALTA valine transaminase [c]: akg + val-L <==>3mob + glu-L VPAMT Valine-pyruvate [c]: 3mob + ala-L --> pyr + val-Laminotransferase

TABLE 13 Metabolite names corresponding to the abbreviations in thereaction equations. Abbreviation Name 1pyr5c 1-Pyrroline-5-carboxylate2ddg6p 2-Dehydro-3-deoxy-D-gluconate 6-phosphate 2pg D-Glycerate2-phosphate 3mob 3-Methyl-2-oxobutanoate 3pg 3-Phospho-D-glycerate 4abut4-Aminobutanoate 6pgc 6-Phospho-D-gluconate 6pgl6-phospho-D-glucono-1,5-lactone ac Acetate accoa Acetyl-CoA acg5saN-Acetyl-L-glutamate 5-semialdehyde adp ADP akg 2-Oxoglutarate ala-DD-Alanine ala-L L-Alanine amp AMP asn-L L-Asparagine asp-L L-Aspartateatp ATP cbp Carbamoyl phosphate cit Citrate co2 CO2 coa Coenzyme A dhapDihydroxyacetone phosphate e4p D-Erythrose 4-phosphate etoh Ethanol f6pD-Fructose 6-phosphate fad FAD fadh2 FADH2 fdp D-Fructose1,6-bisphosphate for Formate fum Fumarate g3p Glyceraldehyde 3-phosphateg6p D-Glucose 6-phosphate glc-D D-Glucose

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1-10. (canceled)
 11. A method for the preparation of 2-hydroxyisobutyricacid (2-HIBA) comprising the successive steps allowing conversion ofacetyl-CoA into 2-hydroxyisobutyric acid, said successive stepsconsisting in: a) converting acetyl-CoA into 3-hydroxybutyryl-CoA b)converting 3-hydroxybutyryl-CoA previously obtained into2-hydroxyisobutyryl-CoA, and c) converting 2-hydroxyisobutyryl-CoA into2-hydroxyisobutyric acid, wherein the steps a), b) and c) are enzymaticconversions.
 12. The method of claim 11, wherein the enzymatic activityin step a) is obtained with the combination of two enzymes, the firstenzyme a1) having an acetoacetyl-CoA thiolase or acetyl-CoAacetyl-transferase activity and the second enzyme a2) having a3-hydroxybutyryl-CoA dehydrogenase activity.
 13. The method of claim 12,wherein enzyme a1) is a gene product encoded by genes selected among thegroup consisting of atoB of E. coli, thlA of C. acetobutylicum and phaAof R. eutropha.
 14. The method of claim 12, wherein enzyme a2) is a geneproduct encoded by genes selected from the group consisting of hbd of C.acetobutylicum and phaB of R. eutropha.
 15. The method of claim 11,wherein step b) is obtained with an enzymatic system having ahydroxyisobutyryl-CoA mutase activity.
 16. The method of claim 15,wherein the hydroxyisobutyryl-CoA mutase activity is performed byenzymes resulting from the gene products encoded by the genes icmA andicmB from A. tertiaricarbonis, from M. petroleiphilum or fromStreptomyces spp.
 17. The method of claim 16 wherein the activity of thehydroxyisobutyryl-CoA mutase is increased by overexpressing the fldA-fpractivation system.
 18. The method of claim 11, wherein step c) isobtained by transfer of CoA on a substrate with an enzyme having a CoAtransferase activity.
 19. The method of claim 18, wherein the enzyme hasan acetyl-CoA transferase activity and the substrates are acetate and2-hydroxyisobutyryl-CoA.
 20. The method of claim 11, wherein step c) isobtained by transfer of CoA on a substrate with an enzyme having anacyl-CoA thioesterase activity.
 21. The method of claim 20, wherein theacyl-CoA thioesterase activity is performed by enzymes resulting fromthe gene products encoded by genes selected from the group consisting oftesB of E. coli and ybgC from H. influenzae.
 22. The method of claim 11,wherein step c) is obtained with the combination of two enzymes thefirst enzyme c1) having a phosphotransacylase activity and the secondenzyme c2) having an acid-kinase activity.
 23. The method of claim 22,wherein enzyme c1) has a phosphate hydroxyisobutyryltransferaseactivity, which is optionally a gene product encoded by gene ptb of C.acetobutylicum.
 24. The method of claim 22, wherein enzyme c2) is ahydroxyisobutyrate kinase, which is optionally a gene product encoded bygene buk of C. acetobutylicum.
 25. The method of claim 11, whereinacetyl-CoA is obtained by bioconversion of a source of carbon in amicroorganism.
 26. The method of claim 11, wherein steps a), b) and c)are performed by a microorganism expressing the genes coding for theenzymes having the enzymatic activities necessary for the conversions ofsaid steps a), b) and c).
 27. The method of claim 11, wherein steps a),b) and c) are performed by the same microorganism.
 28. The method ofclaim 27, wherein the same microorganism provides for the bioconversionof glucose into acetyl-CoA.
 29. A microorganism modified for an improvedproduction of 2-hydroxyisobutyric acid, wherein said microorganismexpresses the genes coding for the enzymes having the enzymaticactivities necessary for the following conversions a) convertingacetyl-CoA into 3-hydroxybutyryl-CoA b) converting 3-hydroxybutyryl-CoApreviously obtained into 2-hydroxyisobutyryl-CoA, and c) converting2-hydroxyisobutyryl-CoA into 2-hydroxyisobutyric acid.
 30. Themicroorganism of claim 29 which is modified to produce higher levels ofacetyl-CoA.
 31. The microorganism of claim 30, wherein the expression ofat least one of the following genes is attenuated: pta encodingphospho-transacetylase ackA encoding acetate kinase poxB encodingpyruvate oxidase ldhA encoding lactate dehydrogenase aceA encodingisocitrate lyase.
 32. The microorganism of claim 29, wherein theavailability of NADPH is increased.
 33. The microorganism of claim 32,wherein the expression of at least one of the following genes isattenuated: pgi encoding the glucose-6-phosphate isomerase udhA encodingthe soluble transhydrogenase.
 34. The microorganism of claim 29comprising a bacteria, optionally selected from the group consisting ofEnterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae andCorynebacteriaceae.
 35. A method for the fermentative production of2-hydroxyisobutyric acid (2-HIBA) by conversion of a simple source ofcarbon into 2-HIBA comprising the steps of: culturing the microorganismof claim 29 in an appropriate culture medium comprising a simple sourceof carbon, and recovering the 2-hydroxyisobutyric acid (2-HIBA) from theculture medium.
 36. The method of claim 35, wherein the2-hydroxyisobutyric acid (2-HIBA) is further purified.